Digital holographic microscope for highly efficient measurement of fluorescent images

Abstract

To provide a digital holographic microscope with which it is possible to acquire a three-dimensional fluorescence distribution by fluorescent holography with high efficiency and using a simple system.SOLUTION: Provided is a digital holographic microscope equipped with a fluorescent holographic optical system that acquires a fluorescence image by fluorescent signal light of an observation object sample, wherein the fluorescent holographic optical system comprises separation means for separating the fluorescent signal light into two polarized lights the polarization directions of which are orthogonal to each other; and an optical system for causing an off-axis hologram to be generated when re-combining the two separated lights of the fluorescent signal light and acquiring a fluorescence image. The separation means uses a polarizing prism utilizing double refraction as it separates the fluorescent signal light into linear polarized lights that are orthogonal to each other, with a Wollaston prism, etc., used.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a digital holographic microscope that acquires three-dimensional fluorescence distribution with high efficiency using fluorescence holography. [Background technology]

[0002] Dynamic 3D imaging of living cells is an important measurement technique in the field of biological applications. 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 to measure changes in cells undergoing mitosis. Various observations become possible by visualizing the interactions between nuclei and proteins in cells stained with fluorescent molecules. Confocal laser scanning microscopes are known as a technique that can measure fluorescent images with high resolution and high contrast. However, confocal laser scanning microscopes require scanning of the focal point for each point on the measurement object, so it takes time to obtain a fluorescent image, and there are inherent limitations to the technique, such as it being unsuitable for measuring phenomena in which dynamic objects change rapidly or for measuring multiple cells at different depths simultaneously. There is also a demand for technology that can simultaneously observe structures such as cell nuclei and cell walls in addition to fluorescent images. Phase contrast microscopes and differential interference microscopes are available as means of structural observation. Phase contrast images are effective for measuring the shape of transparent objects. If fluorescent images and phase images can be observed simultaneously, different information can be obtained at the same time, making it possible to provide more detailed information on bioreactions.

[0003] Digital holographic microscopes are known as a tool that enables instantaneous 3D measurements. In digital holographic microscopes, 3D information is acquired as hologram information, and the light wave information of the object relative to the depth position is reconstructed by performing backpropagation calculations of light waves in a computer. The features of digital holographic microscopes include the ability to observe living cells in 3D without the need for special fluorescent staining, an autofocusing function that allows the focal position to be changed arbitrarily by computer reconstruction, and the ability to perform quantitative phase measurement. Therefore, even in situations where cells move in 3D, a reconstructed image that is automatically in focus can be obtained by the reconstruction calculation.

[0004] When applying digital holographic microscopes to cell observation, it is necessary to obtain holograms of fluorescent information. However, in order to realize holography technology using fluorescence, fluorescent light must be incoherent light, which is highly resistant to interference. In recent years, a number of methods have been proposed for creating holograms from incoherent light, and research into fluorescent digital holographic microscopes is active. In addition, while the fluorescence digital holographic microscope is suitable for targeted measurement, it has the problem that it is ineffective for substances that do not emit fluorescence. In addition, when performing fluorescence observation, the measurement target is limited because harmful fluorescent molecules are used. Therefore, from the perspective of expanding the application of the digital holographic microscope, a multimodal digital holographic microscope that can observe various information such as phase images, fluorescence images, and even polarization is required.

[0005] One of the present inventors has already proposed a digital holographic microscope that combines a fluorescence digital holographic microscope with a phase image detection digital holographic microscope and is capable of measuring fluorescence images and phase images simultaneously (see Patent Documents 1 and 2). However, because digital holographic microscopes use the interference of two light waves, they need to expand the light wave spatially, split it into two light waves, and then superimpose them again, which means that the fluorescent energy needs to be increased in order to observe it with an image sensor. As a result, the light energy irradiated to the observation object increases, and when the observation object is a living cell, phototoxicity that damages the cell becomes a major problem. When applying digital holographic microscopes to the bioimaging field, it is necessary to further improve (highly efficient) the efficiency of light energy utilization in order to avoid this phototoxicity. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Retable 2018 / 070451 [Patent Document 2] Retable 2016 / 163560 Summary of the Invention [Problem to be solved by the invention]

[0007] In view of the above circumstances, the present invention aims to provide a digital holographic microscope that can acquire a three-dimensional fluorescence distribution by fluorescence holography with high efficiency and a simple system. Another object of the present invention is to provide a digital holographic microscope that combines a fluorescence digital holographic microscope with a phase image detection digital holographic microscope, can measure a fluorescence image and a phase image simultaneously, and can measure a fluorescence image with high efficiency. [Means for solving the problem]

[0008] In order to achieve the above object, the digital holographic microscope of the present invention is equipped with a fluorescence holographic optical system that acquires a fluorescent image from fluorescent signal light of an observation sample, the fluorescent holographic optical system comprising a separation means that separates the fluorescent signal light into two polarized lights whose polarization directions are perpendicular to each other, and an optical system that generates an off-axis hologram when the two separated fluorescent signal lights are recombined to acquire a fluorescent image. According to the above configuration, a three-dimensional fluorescence distribution can be obtained by fluorescence holography with high efficiency and in a simple system. Here, the separation means uses a birefringent polarizing element that can separate the light into two polarized lights whose polarization directions are orthogonal to each other. Specifically, a polarizing prism that utilizes birefringence is used to separate the fluorescent signal light into linearly polarized lights that are orthogonal to each other. Examples of polarizing prisms include a Wollaston prism, a Rochon prism, a Nicol prism, and a Glan-Thompson prism, and in particular, a Wollaston prism is preferably used.

[0009] The digital holographic microscope of the present invention may further include a phase image detection holographic optical system that obtains a phase image by interference light obtained by superimposing an object light that passes through a sample to be observed and a reference light that does not pass through the sample, thereby making it possible to simultaneously measure a fluorescent image and a phase image, and to measure the fluorescent image with high efficiency. In the digital holographic microscope of the present invention, the separating means is designed to act only on the fluorescent holographic optical system. The separation means is designed to have no sensitivity to the polarization of the object light of the phase image detection holographic optical system that acquires a phase image and to cause no lens action, while it is designed to have strong sensitivity to the polarization of the fluorescence of the fluorescence holographic optical system that acquires a fluorescent image. As a result, only the polarization of the fluorescence of the fluorescent holographic optical system is separated by the separation means, and an off-axis hologram is generated when the light is combined again.

[0010] In the digital holographic microscope of the present invention, it is preferable that both the fluorescence holographic optical system and the phase image detection holographic optical system are of the reflective type. In a fluorescence holographic optical system that obtains a fluorescence image by using fluorescence excitation light of a sample to be observed, the fluorescence signal is weak, so if it is a transmission type, it is very difficult to cut out only the strong fluorescence excitation light, so a reflection type is preferable. By making both the fluorescence holographic optical system and the phase image detection holographic optical system a reflection type and sharing the optical system, the device can be made more compact. If the fluorescence holographic optical system is a transmission type rather than a reflection type, it is necessary to use a dichroic mirror that sufficiently cuts out the fluorescence excitation light that excites the fluorescence signal and passes only the fluorescence signal, or a bandpass filter that passes a specific wavelength of the fluorescence wavelength and the laser wavelength for phase measurement.

[0011] 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 a fluorescent image and a phase image as holograms. The fluorescent image and the phase image are then separated in the spatial frequency plane, and the object light and the fluorescent signal light are reconstructed from the respective interference intensity distributions. A fluorescent image is obtained as an off-axis hologram when two polarized lights of fluorescent signal light in a fluorescent holographic optical system are combined. A phase image can also be obtained as an off-axis hologram. Both are equi-angle interference patterns of off-axis holograms, but they can be separated in the spatial frequency plane, and the hologram pattern of the fluorescent image and the hologram pattern of the phase image can be separated in the spatial frequency plane from an image captured by an imaging means. By Fourier transforming the two hologram patterns, the components of the equi-angle interference pattern can be extracted in the spatial frequency plane using a bandpass filter.

[0012] The digital holographic microscope of the present invention is a powerful measurement tool in the field of bioimaging, and enables simultaneous and high-speed capture of fluorescent and phase images. In other words, the digital holographic microscope of the present invention can be used to realize a cell observation method that simultaneously obtains spatiotemporal information of a cell nucleus from a fluorescent image and the spatiotemporal structure of the cell nucleus and cell wall from a phase image. Effect of the Invention

[0013] The digital holographic microscope of the present invention has the effect of being able to obtain a three-dimensional fluorescence distribution by fluorescence holography in a highly efficient and simple system, and of being able to solve the phototoxicity problem in the field of bioimaging. In addition, the digital holographic microscope of the present invention combines a fluorescence digital holographic microscope with a phase image detection digital holographic microscope, and has the effect of being able to simultaneously measure a fluorescence image and a phase image, and to measure the fluorescence image with high efficiency. Methods using a diffraction grating, such as the digital holographic microscope already proposed by the inventors, utilize diffraction, making it difficult to achieve a sufficiently high diffraction efficiency for images in which the subject has a complex fluorescence distribution. However, the digital holographic microscope of the present invention does not use a diffractive optical element, so the efficiency of light energy utilization can be increased. [Brief description of the drawings]

[0014] [Figure 1] FIG. 1 is a diagram showing the optical system of the digital holographic microscope according to the first embodiment. [Diagram 2] Illustration of a Wollaston prism [Diagram 3] Illustration of an off-axis hologram produced by a Wollaston prism [Figure 4] Fluorescence hologram and its reconstructed image [Diagram 5] FIG. 1 is a diagram showing the optical system of a digital holographic microscope according to a second embodiment of the present invention. [Figure 6] FIG. 3 is a diagram showing the optical system of the digital holographic microscope according to the third embodiment. [Figure 7] Diagram 1 of the principle of separation and reconstruction of fluorescence holograms and phase holograms [Figure 8] Diagram 2 explaining the principle of separating and reconstructing a fluorescence hologram and a phase hologram [Figure 9] Intensity distribution graphs for the reconstruction propagation distance of the three reconstruction images [Figure 10]Figure showing the results of fluorescence and phase measurements of Physcomitrella patens [Figure 11] Illustration of the optical system of a differential interference microscope using a Wollaston prism BEST MODE FOR CARRYING OUT THEINVENTION

[0015] Hereinafter, an example of an embodiment of the present invention will be described in detail with reference to the drawings. Note that the scope of the present invention is not limited to the following examples and illustrated examples, and many modifications and variations are possible. Example 1

[0016] Figure 1 shows the configuration of an optical system of one embodiment of the digital holographic microscope of the present invention. The digital holographic microscope 101 shown in Figure 1 is a digital holographic microscope equipped with a fluorescence holographic optical system that acquires a fluorescent image using fluorescent signal light of an observation sample, in which the fluorescent holographic optical system includes a separation means that separates the fluorescent signal light into two polarized lights whose polarization directions are perpendicular to each other, and an optical system that generates an off-axis hologram when the two separated fluorescent signal lights are recombined to acquire a fluorescent image.

[0017] The optical system of a digital holographic microscope 101 will be described with reference to Figures 1 to 3. In the digital holographic microscope 101, a laser light source 12 is used to irradiate a measurement object of an observation sample 1 on a sample stage 11 with fluorescence excitation light, thereby exciting fluorescent molecules in the observation sample 1. The digital holographic microscope 101 is a reflection microscope in which illumination light comes from the same side as the objective lens 14, and an image of the sample is observed by the light reflected from the observation sample 1. The excited fluorescent molecules emit long-wavelength fluorescent signal light when irradiated with the fluorescent excitation light, and the long-wavelength fluorescent signal light enters 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, and reflects the fluorescent excitation light and transmits the fluorescent signal light.

[0018] The fluorescence signal light that has passed through the dichroic mirror 13 passes through lens 17a and then polarizer 15. As shown in Fig. 3, the randomly polarized fluorescence signal light 31 is converted by polarizer 15 into linearly polarized light 32 whose vibration direction (vibration plane) vibrates in only one specific direction. After passing through polarizer 15, the fluorescence signal light passes through Wollaston prism 16 and is separated into two linearly polarized light beams (33, 34) whose vibration directions are perpendicular to each other, and travel at different angles. Here, the Wollaston prism is a polarizing prism in which two calcite crystals (3a, 3b) are bonded together so that their optical axes are perpendicular to each other, as shown in Figure 2. Light that is perpendicularly incident on the Wollaston prism 3 becomes two linearly polarized light beams that are perpendicular to each other due to the two bonded prisms, and each beam has approximately equal light intensity.

[0019] The fluorescent signal light that passed through Wollaston prism 16 is collected through lens 17b, and then passes through polarizer 18 to align the polarization, and the two separated orthogonal linearly polarized lights are combined. As shown in Figure 3, the two separated orthogonal linearly polarized lights are slightly angled when combined, resulting in off-axis interference, which produces an off-axis hologram 40 with an equiangular pattern. The imaging system also superimposes the two light waves spatially and temporally. The depth direction can be recorded because the second-order phase term, which is depth information, remains due to interference. In addition, in polarizing prisms that use birefringence, such as Wollaston prisms, the phase difference between the ordinary and extraordinary rays also contributes to the second-order phase term, which is depth information of an object, remaining. By placing a polarizing prism that uses birefringence and an image sensor in an imaging relationship, the two separated orthogonal fluorescent linearly polarized lights completely overlap and form an off-axis hologram with an equi-tilt pattern, resulting in a hologram that is highly efficient and stable in time and space.

[0020] The fluorescent signal light combined 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 bandpass filter 19 passes wavelengths in the fluorescent wavelength band of the fluorescent signal light. The bandpass filter improves the temporal coherence and contrast of the interference fringes, resulting in reduced noise in the reconstructed image and a high-contrast, high-resolution reconstruction.

[0021] The EM-CCD camera 20, which is the imaging means, acquires the fluorescent image as an off-axis hologram of an equiangular pattern, and a computer (not shown) extracts the amplitude distribution and phase distribution of the fluorescent signal light from the hologram of the equiangular interference pattern using the Fourier transform method, and by back-propagating this to the positions of the fluorescent molecules in the sample to be observed using the off-axis method, the wavefront of the fluorescent signal light of the sample to be observed is reproduced and the fluorescent signal light is reconstructed. The EM-CCD camera 20 is equipped with a CCD image sensor with an electron multiplication function, which multiplies the optical signal without increasing noise and enables high-speed imaging even with weak light.

[0022] Figure 4 shows a fluorescence hologram and its reconstructed image measured using the digital holographic microscope of this Example 1. In the fluorescence hologram shown in Figure 4(1), beautiful linear interference fringes were obtained, and separation was good. Example 2

[0023] Fig. 5 shows the configuration of an optical system of another embodiment of the digital holographic microscope of the present invention. The digital holographic microscope 102 shown in Fig. 5 is a digital holographic microscope equipped with a fluorescence holographic optical system for acquiring a fluorescent image by the fluorescent signal light of an observation target sample, similar to the digital holographic microscope 101 of Example 1, in which the fluorescent holographic optical system includes a separation means for separating the fluorescent signal light into two polarized lights whose polarization directions are orthogonal to each other, and an optical system for generating an off-axis hologram when the two separated fluorescent signal lights are combined again to acquire a fluorescent image. The digital holographic microscope 101 of Example 1 is a reflection type (epi-illumination type), whereas the digital holographic microscope 102 of this example is a transmission type. When using a transmissive type, it is necessary to sufficiently cut out the fluorescence excitation light that excites the fluorescence signal light, and so a dichroic mirror that passes only the fluorescence signal light and a bandpass filter that passes specific wavelengths of fluorescent wavelength light are important. Example 3

[0024] 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 further includes a reflection-type phase image detection digital holographic optical system in addition to the digital holographic microscope 101 of Example 1, and in the two optical systems, the fluorescence holographic optical system and the phase image detection holographic optical system, the object light and the fluorescent signal light are transmitted through the objective lens from the sample stage to the imaging means of the image sensor as a common optical path, and are superimposed coaxially, so that two holograms, a phase image and a fluorescent image, can be simultaneously obtained by the common imaging means. The fluorescent holographic optical system and the phase image detection holographic optical system will be described below.

[0025] First, the fluorescence holographic optical system has a configuration similar to that of Example 1, and uses a laser light source 12 to irradiate fluorescence excitation light onto a measurement object of the observation sample 1 on the sample stage 11, thereby exciting fluorescent molecules in the observation sample 1. The excited fluorescent molecules emit fluorescent signal light of a long wavelength when irradiated with the fluorescent 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, and reflects the fluorescent excitation light and transmits the fluorescent signal light. The fluorescent 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. The fluorescent molecules in the observation target sample 1 emit fluorescent signal light of a long wavelength when irradiated with the fluorescent excitation light. The fluorescent signal light proceeds to the dichroic mirror 13 together with the fluorescent excitation light reflected by the mirror 10 on the bottom surface of the glass substrate of the sample stage 11. The dichroic mirror 13 reflects the fluorescent excitation light and transmits the fluorescent signal light, so that the fluorescent excitation light can be sufficiently attenuated and the fluorescent signal light can be emphasized.

[0026] The fluorescence signal light that has passed through dichroic mirror 13 passes through lens 17a, and is then converted into linearly polarized light that oscillates in only one specific direction by polarizer 15. After passing through polarizer 15, the fluorescence signal light passes through Wollaston prism 16, where it is split into two linearly polarized light beams whose oscillation directions are perpendicular to each other. The fluorescent signal light that has passed through Wollaston prism 16 passes through lens 17b and is then collected, and the two separated orthogonal linearly polarized lights are then combined by polarizer 18. When the lights are combined, an off-axis hologram with an equi-tilt angle pattern is generated by off-axis interference. The fluorescence signal light combined by the polarizer 18 then passes through a bandpass filter 19 and is imaged by an EM-CCD camera 20. The EM-CCD camera 20, which serves as imaging means, acquires a fluorescence image as an off-axis hologram of an equiangular pattern, and a computer extracts the amplitude distribution and phase distribution of the fluorescence signal light from the hologram of the equiangular interference pattern using the Fourier transform method, thereby reconstructing the fluorescence signal light.

[0027] On the other hand, in the phase image detection holographic optical system, a laser light source 12 is used to illuminate the measurement object of the observation sample 1 on the sample stage 11, just like the fluorescence holographic optical system. However, the laser light is split by a beam splitter 21 into an object light path that passes through the measurement object and an empty reference light path. The object light that passes through the measurement object is reflected by a mirror 10 below the observation sample 1, enters an objective lens 14, and then proceeds to a dichroic mirror 13. Taking advantage of the fact that the wavelength of the laser light that passes through the measurement object 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 measurement object and transmit the other light. The object light that passes through the measurement object interferes with the reference light by a beam splitter 24. At this time, an off-axis hologram, that is, a hologram of an equal angle interference pattern, is generated by making a slight angle between the object light and the reference light. This is captured by an EM-CCD camera 20. The amplitude distribution and phase distribution of the object light are extracted by using the Fourier transform method from the hologram of the equi-angle interference pattern acquired by the EM-CCD camera 20. In the off-axis method, the wavefront of the object light of the sample to be observed is reproduced by backpropagating it to the original object position, and the object light can be reconstructed.

[0028] Here, the principle of separating and reproducing a fluorescence hologram and a phase hologram will be described with reference to Figs. 7 and 8. In the digital holographic microscope of this embodiment, a fluorescence hologram and a phase hologram are acquired by a shared EM-CCD camera 20. Both the fluorescence hologram and the phase hologram are holograms of an equiangular interference pattern. However, by performing a Fourier transform on the two patterns, the phase hologram and the fluorescence hologram, it is possible to extract the equiangular interference pattern components of a signal that exists at a position shifted from the origin in the spatial frequency plane using a window function filter. Specifically, a window function is used to separate two holograms, the fluorescence hologram that appears at different positions in the spatial frequency plane, and the phase hologram that exists at a position shifted from the origin.

[0029] Using the digital holographic microscope of this example, we demonstrated that a sample consisting of fluorescent beads with a diameter of 10 μm placed on a glass slide can be recorded and reproduced in the depth direction by moving the sample in the height direction. Figure 9 shows the reconstruction propagation distance at which 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). From Figure 9, it can be seen that the digital holographic microscope of this embodiment is capable of recording and reproducing in the depth direction from -30 μm to +10 μm. Although the depth position and the reproduction distance are not in a proportional relationship, it can be seen that the depth position can be determined from the reconstruction distance, making three-dimensional measurement possible.

[0030] Next, we demonstrated that fluorescent 3D imaging of living plant cells is possible using Physcomitrella patens introduced with fluorescent proteins. Figure 10 shows the results of fluorescence and phase measurements of Physcomitrella patens introduced with fluorescent proteins, using the digital holographic microscope of this example. Figure 10(1) shows the reconstructed images from z=-10μm to z=20μm in a fluorescent hologram. The imaging of the cell nucleus, which was in focus at the arrow, is reproduced. Figure 10(2) shows the phase distribution measured simultaneously with the measurement of the fluorescent hologram. Comparing z=-5μm and z=20μm, at z=-5μm, the edges are clearly visible and the periphery is in focus. At z=20μm, the structure of the chloroplast near the center could be clearly observed. Since different structures such as the cell nucleus, cell wall, and chloroplast can be observed using fluorescence and phase, respectively, the simultaneous measurement of the fluorescence image and phase image shows that more information on the sample being observed can be obtained.

[0031] FIG. 11 is a schematic diagram of the optical path explaining the principle of differential interference observation. In differential interference observation, when light passes through an observation sample, the difference in the refractive index and thickness of the part of the sample through which the light passes causes a difference in the distance the light travels (optical path difference), and this optical path difference is used to observe transparent samples. A differential interference microscope can capture the minute refractive index difference inside a cell to create a light-dark contrast. 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 a polarizer 51 and becomes linearly polarized light. The light is separated by the Wollaston prism 52 into two linearly polarized lights that are orthogonal to each other and travel at different angles. The light is made parallel to the optical axis by the lens 53 and passes through a glass substrate 54 and an observation target sample 55. At this time, the two linearly polarized lights pass through positions that are spatially separated by the action of the Wollaston prism 52. The two polarized lights are made parallel to the optical axis by the lens 56 and Wollaston prism 57, and by passing through the polarizer 58, are converted into interference intensities having a phase difference at spatially separated positions.

[0032] On the other hand, in the digital holographic microscope of the present invention, a Wollaston prism is used to separate the light into two mutually orthogonal polarized lights with different angles, and an imaging lens is used to overlap the two light waves spatially and temporally. Also, the light is passed through a polarizer to align the polarized light, forming equiangular interference fringes, and the objectives (phase difference and polarization recording) and means are different from those of a differential interference microscope. [Industrial Applicability]

[0033] The present invention is useful for microscopes in the field of bioimaging. [Explanation of symbols]

[0034] 101,102,103 Digital holographic microscope 1,55 Observation sample 11 Sample stage 12 Laser light source 13 Dichroic mirror 14 Objective Lens 15,18 Polarizer 3,16 Wollaston Prism 19 Bandpass Filters 20 EM-CCD camera 17a~17c Lens 10,20,23 Mirror 21,24 Beam splitter 40 Off-Axis Hologram

Claims

1. 1. A digital holographic microscope having a fluorescence holographic optical system for acquiring a fluorescence image of an observation target sample using fluorescence signal light, The fluorescent holographic optical system is a digital holographic microscope characterized in that it comprises: a separation means for separating the fluorescent signal light into two polarized lights whose polarization directions are perpendicular to each other; and an optical system for generating an off-axis hologram when the two separated fluorescent signal lights are recombined to obtain the fluorescent image.

2. 2. The digital holographic microscope according to claim 1, wherein the optical system for acquiring the fluorescent image constitutes an imaging system.

3. 3. The digital holographic microscope according to claim 1, wherein the splitting means splits the fluorescent signal light into linearly polarized lights that are orthogonal to each other using a polarizing prism that utilizes birefringence.

4. 4. The digital holographic microscope according to claim 3, wherein the polarizing prism and an image sensor that captures the fluorescent image are in an image-forming relationship.

5. 5. The digital holographic microscope according to claim 3, wherein the polarizing prism is any one of a Wollaston prism, a Rochon prism, a Nicol prism, and a Glan-Thompson prism.

6. A digital holographic microscope as described in any one of claims 1 to 5, further comprising a phase image detection holographic optical system that acquires a phase image by interference light obtained by superimposing an object light that passes through the observation sample and a reference light that does not pass through the sample.

7. 7. The digital holographic microscope according to claim 6, wherein the separating means acts only on the fluorescent holographic optical system.

8. an imaging means for the fluorescence holographic optical system and an imaging means for the phase image detection holographic optical system are shared; The digital holographic microscope according to claim 6, characterized in that the imaging means simultaneously acquires 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 reconstructs the object light and the fluorescent signal light from their respective interference intensity distributions.

9. 7. The digital holographic microscope according to claim 6, wherein the fluorescence holographic optical system and the phase image detection holographic optical system are both of a reflection type.

Images