JP2015172675A - Observation device, observation method, and illumination device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To acquire three-dimensional image structure data of an observation area without point scanning or Z scanning.SOLUTION: An observation device includes: an illumination section which illuminates an observation area with a three-dimensional stripe pattern having different stripe pitches in a depth direction of the observation area; an observation section which generates an image of the observation area illuminated with the stripe pattern; a scanning section which scans the observation area with the stripe pattern; and a separation section which separates image components on different depths of the observation area, from each other, on the basis of the images generated in different scanning positions.

Description

  The present invention relates to an observation device, an observation method, and an illumination device.

  Conventionally, a point scan type or Z scan type microscope is known as a microscope for observing the three-dimensional structure of a sample with a microscope.

JP 2004-212600 A

  The present invention proposes an observation apparatus, an observation method, and an illumination apparatus suitable for acquiring three-dimensional image structure data of an observation target region without performing point scan or Z scan.

  An aspect of the observation apparatus illustrating the present invention includes an illumination unit that illuminates the observation target region with a three-dimensional fringe pattern having different stripe pitches in the depth direction of the observation target region, and the observation illuminated with the stripe pattern. Based on a plurality of images generated at different scanning positions, an observation unit that generates an image of the target region, a scanning unit that scans the observation target region with the stripe pattern, and different depths of the observation target region And a separation unit that separates a plurality of image components from each other.

  An aspect of the observation method illustrating the present invention includes a procedure for illuminating the observation target region with a three-dimensional stripe pattern having a different stripe pitch in the depth direction of the observation target region, and the observation target illuminated with the stripe pattern. Based on a procedure for generating an image of the region, a procedure for scanning the observation target region with the stripe pattern, and a plurality of the images generated at different scanning positions, a plurality of depths relating to different depths of the observation target region Separating image components from each other.

  One aspect of the illumination device illustrating the present invention illuminates the observation target region with a three-dimensional fringe pattern having different stripe pitches in the depth direction of the observation target region.

  According to the present invention, an observation apparatus, an observation method, and an illumination apparatus suitable for acquiring three-dimensional image structure data of an observation target region without performing point scanning or Z scanning are proposed.

It is a block diagram of 3D illumination microscope apparatus. It is a figure which shows the slit mask. It is a figure explaining observation object surface 5-1, 5-2, and 5-3. It is a schematic diagram of a fringe pattern _{F 1, F 2, F 3} . N images I ₁ , I ₂ ,. It is a conceptual diagram of a I _N. It is a conceptual diagram of the luminance change waveform of the pixel of interest. It is a conceptual diagram of the Fourier spectrum of a luminance change waveform. It is a conceptual diagram of the density distribution data of the fluorescent substance in the pixel of interest. It is a figure explaining fringe pitch P (x, z) in coordinates (x, z). It is a fringe frequency characteristic curve showing the relationship between the depth z and the fringe frequency f. It is a quantized fringe frequency characteristic curve.

[Embodiment]
Hereinafter, a 3D illumination microscope apparatus will be described as an embodiment of the present invention.

  FIG. 1 is a configuration diagram of a 3D illumination microscope apparatus. As shown in FIG. 1, the 3D illumination microscope apparatus 1 includes a laser light source 101, an optical fiber 11, a collimating lens 12, a one-dimensional diffraction grating 13, a cylindrical lens 16, a slit mask 18, and a relay lens. Lenses 25 and 27, a sample 5, a dichroic mirror 28, an imaging lens 29, an image sensor 35, a sample stage 13A, a control device 39, and an arithmetic device 40 are arranged.

  Hereinafter, the entire output end of the optical fiber 11, the collimating lens 12, the one-dimensional diffraction grating 13, the cylindrical lens 16, the slit mask 18, the lens 25, the dichroic mirror 28, and the lens 27 are referred to as “illumination optical system 10”. The entire lens 27, dichroic mirror 28, and imaging lens 29 are referred to as “observation optical system 20”. These illumination optical system 10 and observation optical system 20 share a lens 27 and a dichroic mirror 28.

  The laser light source 101 is a light source having coherence. The emission wavelength of the laser light source 101 is set to be the same as the excitation wavelength of the fluorescent material contained in the sample 5. When the laser light emitted from the laser light source 101 propagates through the optical fiber 11, a point light source is formed at the emission end of the optical fiber 11.

The laser light emitted from the point light source becomes a parallel light beam having a large diameter by the collimator lens 12 and enters the one-dimensional diffraction grating 13. The parallel light beam incident on the one-dimensional diffraction grating 13 is branched into a plurality of diffracted lights having different orders by the diffraction action of the one-dimensional diffraction grating 13.
Hereinafter, the grating line direction of the one-dimensional diffraction grating 13 is defined as a predetermined direction (y direction) perpendicular to the optical axis direction (z direction) of the illumination optical system 10. In this case, the branch direction of the plurality of diffracted lights is a direction (x direction) perpendicular to both the z direction and the y direction.

The plurality of diffracted lights branched by the one-dimensional diffraction grating 13 are subjected to the condensing action of the cylindrical lens 16 to form condensing lines (line light sources) at different positions on the focal plane 6A of the cylindrical lens 16.
Here, the generatrix direction of the lens surface of the cylindrical lens 16 is set to be the same as the grating line direction (y direction) of the one-dimensional diffraction grating 13. In this case, the longitudinal direction of the line light source formed on the focal plane 6A is the y direction, and the arrangement direction of the plurality of line light sources individually formed by the plurality of diffracted lights is the x direction. In FIG. 1, only the 0th-order diffracted light and the ± 1st-order diffracted light among the plurality of diffracted lights are shown.

The plurality of diffracted lights directed toward the focal plane 6A are incident on the slit mask 18 disposed in the vicinity of the focal plane 6A. A pair of slits 18a and 18b is formed in the slit mask 18 as shown in FIG. The longitudinal direction of each of the pair of slits 18a and 18b is the y direction, and the arrangement direction of the pair of slits 18a and 18b is the x direction. Further, the center distance between the pair of slits 18a and 18b is equal to the distance between the pair of line light sources formed by ± 1st order diffracted light individually. The width in the short direction of each of the pair of slits 18a and 18b is equal to the width in the short direction of each of the pair of line light sources (determined by the size of the point light source described above).
As shown in FIG. 1, among the plurality of diffracted lights incident on the slit mask 18, 0th-order diffracted light and second-order and higher-order diffracted lights are shielded by the light shielding portion of the slit mask 18, and ± 1st-order diffracted lights are The pair of slits 18a and 18b are individually passed. Each of the ± 1st-order diffracted lights individually passing through the pair of slits 18a and 18b is a cylindrical wave, and the generatrix direction of the wave front of these cylindrical waves is the y direction. That is, a pair of line light sources is individually formed on the lens 25 side of the pair of slits 18a and 18b. The spread angle of a pair of cylindrical waves emitted individually from a pair of line light sources is not necessarily the same as the angle shown in FIG.

  When a pair of cylindrical waves individually emitted from a pair of slits 18a and 18b (a pair of line light sources) pass through the lens 25, the dichroic mirror 28, and the lens 27 in this order, they are conjugate with the focal plane 6A with respect to the lenses 25 and 27. A pair of line light sources 18a 'and 18b' are formed on the flat surface 6A '.

  Of the pair of line light sources 18a ′ and 18b ′, the line light source 18a ′ is an image of the line light source (secondary line light source) formed on the lens 25 side of the slit 18a, and the line light source 18b ′ is the slit 18b. It is the image (secondary line light source) of the line light source formed in the lens 25 side.

  A pair of cylindrical waves are individually emitted from the pair of secondary line light sources 18a 'and 18b'. The generatrix direction of the wave front of the pair of cylindrical waves is the y direction.

  A pair of cylindrical waves individually emitted from the pair of secondary line light sources 18 a ′ and 18 b ′ interfere with each other in the vicinity of the sample 5 to form a three-dimensional interference fringe. Since the generatrix direction of the wave fronts of the pair of cylindrical waves is the y direction, the interference fringes formed in the vicinity of the sample 5 have an intensity distribution in the x direction and the z direction. The intensity of is uniform.

  According to such interference fringes, a plurality of fringe patterns having different fringe pitches are individually formed on the plurality of xy cross sections having different z coordinates in the sample 5.

  Hereinafter, attention is focused on the observation target region 50 of the sample 5. The “observation target region 50” is a region within the field of view of the observation optical system 20 and within the depth of focus of the observation optical system 20. Further, on behalf of various xy cross sections of the observation target region 50, assuming three observation target surfaces 5-1, 5-2, and 5-3 as shown in FIG. Ignore other xy sections.

Among these, the z coordinate z ₂ of the observation target surface 5-2 is located on the focal plane of the observation optical system 20, and the z coordinate z ₁ of the observation target surface 5-1 is the coordinate viewed from the observation optical system 20 side. located deeper than z _2, z coordinate z ₃ of the observation target surface 5-3 is located in front of the coordinate z ₂ as viewed from the side of the observation optical system 20.

Therefore, as shown in FIG. 3B, the spatial frequency (hereinafter referred to as “fringe frequency”) f _{1 of} the fringe pattern F ₁ formed on the observation target surface 5-1 is formed on the observation target surface 5-2. that fringe frequency _{f 2} of the fringe pattern _{F 2,} the relationship of the fringe frequency _{f 3} of the fringe pattern _{F 3} which is formed on the observation target plane _5-3, the f _{1 <f} 2 _{<f 3.}

4A to 4C are schematic diagrams of the fringe patterns F ₁ , F ₂ , and F ₃ viewed from the observation optical system 20, and FIG. 4D is viewed from the observation optical system 20. it is a schematic view of the entire fringe pattern _{F 1, F 2, F 3} .
Returning to FIG. 1, the sample 5 includes biological cells labeled with a fluorescent substance. This fluorescent material emits fluorescence when illuminated with interference fringes. The intensity distribution of the fluorescence emitted from the observation target region 50 is determined according to both the three-dimensional density distribution of the fluorescent material existing in the observation target region 50 and the three-dimensional intensity distribution of the interference fringes that illuminate the observation target region 50. .

  The fluorescence emitted from the observation target region 50 enters the beam splitter 28 via the lens 27 and reflects the beam splitter 28. When the fluorescence reflected by the beam splitter 28 passes through the imaging lens 29, a fluorescence image of the observation target region 50 is formed on the imaging surface of the imaging element 35.

Here, the imaging surface of the imaging device 35 is conjugate with the observation target surface 5-2 (see FIG. 3) with respect to the observation optical system 20, and the observation target surfaces 5-1 and 5-3 are those of the observation optical system 20. It is within the depth of focus. Therefore, all of the fringe patterns F ₁ , F ₂ , and F ₃ shown in FIG. 3 appear in the fluorescent image on the imaging surface without blurring.

  When the image sensor 35 generates an image by capturing a fluorescent image on the imaging surface, the image sensor 35 outputs the image to the control device 39.

  Here, the sample 5 can be shifted at least in the x direction by the sample stage 13A. According to this shift, the observation target region 50 is scanned in the x direction by the interference fringes (stripe scanning).

Here, the sample 5 is shifted in order to scan the observation target region 50 in a fringe pattern, but interference fringes may be shifted instead of the sample 5. In order to shift the interference fringes, for example, the entire illumination optical system 10 may be shifted in the x direction.
The control device 39 drives the sample stage 13A to scan the sample 5 in the x direction at a regular pitch, and drives the imaging device 35 when the stripe scanning position (stripe scanning position) is at each position. When N images are acquired, the N images are output to the arithmetic device 40. The N images have different fringe scanning positions, but the fringe scanning pitch is uniform among the N images.

  The calculation device 40 performs a calculation process (described later) on the N images transferred from the control device 39, thereby calculating a three-dimensional density distribution of the fluorescent substance existing in the observation target region 50, and the three-dimensional density distribution. Is displayed on an image display device (not shown).

  Next, arithmetic processing by the arithmetic device 40 will be described in detail.

FIG. 5 shows N images I ₁ , I ₂ ,. It is a conceptual diagram of a I _N. N images I ₁ , I ₂ ,. The stripe scanning positions S are different between I _N and N images I ₁ , I ₂ ,. Image _I 1 frame number of N sheets of _{I N,} I _2, .... It represents the fringe scanning position S of I _N. Therefore, if the shift of the fringe scanning position S between two adjacent images I _i and I _{(i + 1) of} the frame number is ΔS, the fringe scanning position S _i of the image I _i of the i-th frame is S _i = ( N−1) × ΔS. These N images I ₁ , I ₂ ,. For I _N, arithmetic unit 40 performs the following steps (1) to (4).

(1) The arithmetic unit 40 has N images I ₁ , I ₂ ,. I _N (N) pixel values I ₁ (x, y), I ₂ (x, y),. Reference is made to I _N (x, y). N pixel values I ₁ (x, y), I ₂ (x, y),. When I _N (x, y) is plotted in the order of frame numbers, for example, a luminance change waveform as shown in FIG. 6 can be drawn. The horizontal axis in FIG. 6 indicates the frame number (that is, the fringe scanning position S), and the vertical axis in FIG. 6 indicates the pixel value (luminance value). Note that the luminance change waveform in FIG. 6 is a simplified version of the actual luminance change waveform, and the actual luminance change waveform includes more frequency components than the luminance change waveform in FIG.

The luminance change waveform in FIG. 6 includes the luminance component (luminance component indicating the density of the fluorescent material at z = z ₁ ) related to the observation target surface 5-1 in FIG. (A luminance component indicating the density of the fluorescent material at z = z ₂ ) and a luminance component related to the observation target surface 5-3 in FIG. 3 (a luminance component indicating the density of the fluorescent material at z = z ₃ ) are included. .

Observation target surface 5-1 of these, FIG. 3, in the fringe scanning are scanned by fringe pattern F ₁ fringe frequency f _1. Therefore, the luminance component that changes at the frequency f ₁ in the luminance change waveform in FIG. 6 is the luminance component related to the observation target surface 5-1.

Moreover, the observation target plane 5-2 in Figure 3, during fringe scanning are scanned by fringe pattern _{F 2} fringes frequency _{f 2.} Therefore, among the luminance change waveform of FIG. 6, the luminance component varying at the frequency f ₂ is a luminance component related to the observation target plane 5-2.

Moreover, the observation target plane 5-3 in Figure 3, during fringe scanning are scanned by fringe pattern _{F 3} of fringe frequency _{f 3.} Therefore, among the luminance change waveform of FIG. 6, the luminance component varying at the frequency f ₃ is a luminance component related to the observation target plane 5-3.

(2) The arithmetic unit 40 operates on the luminance change waveform shown in FIG. 6 (that is, N pixel values I ₁ (x, y), I ₂ (x, y),... I _N (x, y)). For example, a Fourier spectrum as shown in FIG. 7 is acquired by performing Fourier transform in the S direction. However, in FIG. 7, it is assumed that the fluorescent substance is not present in the xy cross section other than the observation target surfaces 5-1, 5-2, and 5-3 in the observation target region 50.

In the Fourier spectrum of FIG. 7, the height of the peak that appears at the frequency f ₁ indicates the luminance component (the density of the fluorescent material at z = z ₁ ) related to the observation target surface 5-1.

Further, in the Fourier spectrum of FIG. 7, the height of the peak that appears at the frequency f ₂ indicates the luminance component (the density of the fluorescent material at z = z ₂ ) related to the observation target surface 5-2.

Further, in the Fourier spectrum of FIG. 7, the height of the peak appearing at the frequency f ₃ indicates the luminance component (the density of the fluorescent material at z = z ₃ ) related to the observation target surface 5-3.

  In the Fourier spectrum of FIG. 7, the height of the peak that appears at frequency 0 indicates the base luminance (DC component) of the interference fringes.

Therefore, according to the Fourier transform of the procedure (2), the DC component, the luminance component related to the observation target surface 5-1 (the density of the fluorescent material at z = z ₁ ), and the luminance component related to the observation target surface 5-2 (z = the density of fluorescent materials) in z _2, the density of the phosphor in the luminance component (z = z ₃ regarding observation target surface 5-3) and are separated from each other.

  (3) The arithmetic unit 40 eliminates the peak appearing at the frequency 0 from the Fourier spectrum of FIG. 7, and coordinates the frequency axis f of the Fourier spectrum to the depth axis z of the sample 5, thereby paying attention. The fluorescent substance density distribution data (see FIG. 8) in the depth direction of the coordinates (x, y) is acquired.

Note that the coordinate transformation in the procedure (3) is a coordinate transformation in which the frequency f ₁ is transformed into the coordinate z ₁ , the frequency f ₂ is transformed into the coordinate z ₂ , and the frequency f ₃ is transformed into the coordinate z _3. .

  (4) The computing device 40 performs the steps (1) to (3) for all the coordinates of interest (x, y), so that the density distribution data in the depth direction at all the coordinates of interest (x, y) ( (See FIG. 8). As a result, the three-dimensional density distribution of the fluorescent material in the entire observation target region 50 is obtained (procedure (4) above).

  Therefore, according to the 3D illumination microscope apparatus 1 of the present embodiment, the three-dimensional structure of the observation target region 50 can be observed without performing a point scan or a Z scan.

  In addition, if a low-magnification imaging optical system having a long focal depth is used as the observation optical system 20 of the present embodiment, a large width (image depth) in the z direction of the observation target region 50 can be secured.

In the above description, as shown in FIGS. 4A, 4 </ b> B, and 4 </ b> C, the fringe frequencies f ₁ , f ₂ , and f ₃ of the fringe patterns F ₁ , F ₂ , and F ₃ are expressed as follows: Although assumed uniform in the x direction, it is actually non-uniform in the x direction.

  Hereinafter, what value the fringe frequency f (x, z) in the coordinates (x, z) of the interference fringe will be described.

  Here, the fringe frequency f (x, z) at the coordinates (x, z) will be described by the fringe pitch P (x, z) at the coordinates (x, z). The fringe pitch P (x, z) is the reciprocal of the fringe frequency f (x, z).

  As shown in FIG. 9, when the center of the pair of secondary line light sources 18a ′ and 18b ′ is the origin of the xz coordinate and the distance between the pair of secondary line light sources 18a ′ and 18b ′ is d, the coordinate (x , Z), the fringe pitch P (x, z) is expressed by the following equation (1).

Therefore, for example, the stripe pitch P (x, z ₁ ) on the observation target surface 5-1 can be obtained by applying z = z ₁ to the equation (1).

Further, for example, the stripe pitch P (x, z ₂ ) on the observation target surface 5-2 can be obtained by applying z = z ₂ to the equation (1).

Further, for example, the stripe pitch P (x, z ₃ ) on the observation target surface 5-3 can be obtained by applying z = z ₃ to the equation (1).

  Next, the z resolution of the 3D illumination microscope apparatus 1 will be described.

  In order to increase the z resolution of the 3D illumination microscope apparatus 1, it is only necessary to narrow the interval between two observation target surfaces whose stripe pattern difference is one or more. For that purpose, it is only necessary to generate an interference fringe whose fringe frequency changes greatly in the z direction.

  On the other hand, in order to separate the luminance components of two adjacent observation target surfaces from each other, it is necessary to increase the resolution Δf in the f-axis direction of the Fourier spectrum (see FIG. 7). For this purpose, a large data range (sampling range) T in the S-axis direction of the luminance change waveform (see FIG. 6) should be ensured (because the relationship of Δf = 1 / (2T) is established).

  Based on the above, a numerical example of the 3D illumination microscope apparatus 1 will be described below.

・ Light source wavelength λ: 600 nm
-Space d between secondary line light sources 18a 'and 18b': 0.1 mm
-Refractive index n of the medium: 1.33
The xy cross-sectional size of the observation target region 50 (= the visual field range of the observation optical system 20 in which the stripe pitch can be regarded as uniform): 400 μm × 400 μm
FIG. 10 is a characteristic curve (fringe frequency characteristic curve) showing the relationship between the depth z of the sample 5 and the fringe frequency f in this numerical example. This characteristic curve is calculated based on the equation (1).

  As shown in FIG. 10, in the z coordinate range A where z = 1 to z = 1.5 [mm], the change amount of the fringe frequency f with respect to the change amount of the depth z is relatively large, whereas z = 2 to 2. In the z coordinate range B of 2.5 [mm], the change amount of the fringe frequency f with respect to the change amount of the depth z is relatively small. That is, in the interference fringes, the closer to the secondary line light sources 18a 'and 18b', the higher the possibility of realizing a high z resolution.

  FIG. 11A shows a fringe frequency characteristic curve in the z coordinate range A, and FIG. 11B shows a fringe frequency characteristic curve in the z coordinate range B. Note that the data shown in FIGS. 11A and 11B is quantized data. In this quantization, it is assumed that the value of Δf determined by the data range T is exactly 1 (lines / mm).

  As shown in FIG. 11 (A), in the z coordinate range A, the interval between two adjacent observation target surfaces, that is, the z resolution Δz is about 20 μm, and as shown in FIG. 11 (B), the z coordinate range B It can be seen that the interval between two observation target surfaces adjacent to each other, that is, the z resolution Δz is about 50 μm.

  Of course, if the data range T is increased and the value of Δf is decreased (the resolution is improved), Δz is further improved.

[Supplement of embodiment]
In the present embodiment, the type of the illumination optical system 10 of the 3D illumination microscope apparatus 1 is epi-illumination, but transmission illumination may be used. In that case, the beam splitter 28 becomes unnecessary. Also, the relay lenses 25 and 27 are not essential.

  In the present embodiment, the light incident on the pair of slits 18a and 18b is a condensed light flux, so that the light quantity loss in the slit mask 18 can be minimized. However, when the light quantity loss in the slit mask 18 can be tolerated, the light incident on the pair of slits 18a and 18b may be a parallel light beam instead of a condensed light beam.

  In the present embodiment, a pair of diverging cylindrical waves are used to generate interference fringes having different fringe frequencies in the depth direction of the sample 5, but a pair of converging cylindrical waves is also used. Good.

  In the present embodiment, a pair of diverging cylindrical waves are used to generate interference fringes having different fringe frequencies in the depth direction of the sample 5, but a pair of converging spherical waves or a pair of diverging waves. A spherical wave may be used.

  In the present embodiment, the interference fringes are generated in order to illuminate the sample 5 with a three-dimensional fringe pattern having different fringe frequencies in the depth direction of the sample 5, but instead of generating the interference fringes, two-dimensional fringes are generated. An image of the mask on which the fringe pattern is displayed may be projected onto the sample. For example, if an image of a mask on which a fringe pattern is displayed is projected onto the sample 5 by non-parallel illumination light (divergent or convergent light), the sample has a three-dimensional fringe pattern having different fringe frequencies in the depth direction of the sample 5. 5 can be illuminated. Note that a transmissive or reflective liquid crystal display element or the like can be used as the mask.

[Effects of Embodiment]
The observation apparatus (3D illumination microscope apparatus 1) of the present embodiment has a three-dimensional fringe pattern (interference fringes) with different fringe pitches in the depth direction (z direction) of the observation target area (50). ) For illuminating the observation target region illuminated by the fringe pattern (interference fringes) (observation optical system 20, imaging element 35), and the observation Based on the scanning unit (sample stage 13A, control device 39) that scans the target area (50) with the fringe pattern (interference fringe) and the plurality of images generated at different scanning positions, the observation target area (50 ) Are separated from each other by a plurality of image components (luminance components) relating to different depths.

  Therefore, the observation apparatus (3D illumination microscope apparatus 1) of the present embodiment can observe the three-dimensional structure of the observation target region (50) without performing point scanning or Z scanning.

  The illumination unit (illumination optical system 10) generates the fringe pattern (interference fringes) by causing a pair of coherent non-parallel lights to interfere with each other in the observation target region (50).

  The illumination unit (illumination optical system 10) generates the pair of non-parallel light by a slit mask (18) having a pair of slits (18a, 18b).

  In addition, the separation unit (arithmetic device 40) performs the separation by performing Fourier transform on the plurality of images in the scanning direction.

  DESCRIPTION OF SYMBOLS 1 ... 3D illumination microscope apparatus, 101 ... Laser light source, 11 ... Optical fiber, 12 ... Collimating lens, 13 ... One-dimensional diffraction grating, 16 ... Cylindrical lens, 18 ... Slit mask, 25, 27 ... Lens, 5 ... Sample, 28 ... Dichroic mirror, 29 ... Imaging lens, 35 ... Imaging element, 13A ... Sample stage, 39 ... Controller, 40 ... Arithmetic unit

Claims (6)

An illumination unit that illuminates the observation target region with a three-dimensional fringe pattern having different stripe pitches in the depth direction of the observation target region;
An observation unit for generating an image of the observation target region illuminated with the stripe pattern;
A scanning unit that scans the observation target region with the stripe pattern;
An observation apparatus comprising: a separation unit that separates a plurality of image components related to different depths of the observation target region from each other based on the plurality of images generated at different scanning positions. The observation apparatus according to claim 1,
The illumination unit is
The said fringe pattern is produced | generated by making a pair of coherent non-parallel light interfere with each other in the said observation object area | region. The observation apparatus characterized by the above-mentioned. The observation apparatus according to claim 2,
The illumination unit is
The observation apparatus, wherein the pair of non-parallel light is generated by a slit mask having a pair of slits. In the observation apparatus as described in any one of Claims 1-3,
The separation unit is
An observation apparatus, wherein the separation is performed by Fourier transforming a plurality of the images in a scanning direction. Illuminating the observation area with a three-dimensional fringe pattern having different fringe pitches in the depth direction of the observation area;
Generating an image of the observation target area illuminated with the stripe pattern;
A procedure for scanning the observation target area with the stripe pattern;
And a step of separating a plurality of image components relating to different depths of the observation target region from each other based on the plurality of images generated at different scanning positions.   An illumination device that illuminates the observation target region with a three-dimensional fringe pattern having different stripe pitches in the depth direction of the observation target region.

Images