Transmission type optical microscope

Abstract

To provide a transmission type optical microscope having tomographic observation functions.SOLUTION: A transmission type optical microscope 100 comprises: a projector 10 emitting light in a prescribed pattern; a condenser lens 30 configured to condense light so as to form a focal plane F in an observation space in which an object is placed; an objective lens 40 configured to condense light transmitting through the observation space; a light receiving element 51 configured to convert light condensed on the objective lens 40 into an electrical signal; and a signal processing part 60 configured to process the electrical signal for imaging. In a three-dimensional observation space composed of xyz orthogonal coordinate system, the projector 10 displaces in an x direction, fringe pattern light with brightness that is uniform in a y direction but changes periodically in the x direction on the focal plane F, and emits the light in a plurality of patterns. The signal processing part 60 selectively acquires an object at a desired depth in a z direction to the focal plane F on the basis of electrical signals converted from the light in the plurality of patterns.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a transmission optical microscope. [Background technology]

[0002] A transmission optical microscope is a microscope that transmits light through a sample to allow magnified observation. Transmission optical microscopes are suitable for observing thin, transparent samples and are widely used for observing biological samples (see, for example, Patent Document 1). The basic components of a transmission optical microscope are a light source, a lens, a sample, an objective lens, and an eyepiece. Light from the light source is shaped by the lens into parallel light and passes through the sample. The light that has passed through the sample is focused into an image by the objective lens and finally enters the observer's eye through the eyepiece. Furthermore, by providing a light-receiving element (optical sensor) instead of the eyepiece, it is possible to convert the light that has passed through the sample into an electrical signal and generate an image. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] International Publication No. 2019 / 039581 [Non-patent literature]

[0004] [Non-Patent Document 1] M. Neil et al., Optics Letters, December 15, 1997, p.1905, Method of obtaining optical sectioning by using structured light in a conventional microscope [Non-patent document 2] Fukano and Miyawaki, Optics, December 2004, Vol. 33, No. 12, p. 719, Fluorescence microscope using a digital micromirror device [Non-patent document 3] Shigeki Nonaka, Light Sheet Microscopy: A New Microscopic Method for Observing Living Spheres, Microscope, Vol. 47, No. 3, March 2012, p. 162, Light Sheet Microscopy: A New Microscopic Method for Observing Living Spheres [Non-patent document 4] S. Inoue, KR Spring, Translated by Terakawa, Ichie, and Watanabe, Video Microscopy, Kyoritsu Shuppan, pp.108-114 Summary of the Invention [Problem to be solved by the invention]

[0005] It is well known that a microscope would be desirable if it were equipped with a tomographic observation function that allows for hierarchical observation of the observation space in which the object is placed along the optical axis. However, conventional transmission optical microscopes have a problem in that the observation depth of the sample is shallow, making it difficult to obtain information about the depth direction (vertical axis direction) of the sample. That is, with a typical transmission optical microscope, the image of a sample closer to the focal plane is clearer, while the image of a sample farther from the focal plane is blurred. The degree of this blur provides a clue to the approximate position of the sample in the depth direction. Thus, with a typical transmission optical microscope, the visibility of the microstructure in the observed image (the presence or absence and size of high spatial frequency components) provides a clue to obtaining information about the depth direction, but it is difficult to automatically acquire and record information about the depth direction.

[0006] The specimen to be observed appears differently depending on its type and the imaging method of the microscope. When observing a collection of such diverse specimens, it is desirable to know the front-to-back relationship in the depth direction, or to extract only the light intensity within the slice by slicing the observation space containing the specimens perpendicular to the optical axis. Generally speaking, confocal microscopes and ribbon beam microscopes are widely used for such cross-sectional observation, but their application range is limited to highly scattering or fluorescent specimens (Non-Patent Documents 1-4). However, there has not been any known means of implementing such cross-sectional observation capabilities in a transmission optical microscope.

[0007] SUMMARY OF THE INVENTION Therefore, a main object of the present invention is to provide a transmission optical microscope having a tomographic observation function. [Means for solving the problem]

[0008] The present invention relates to a transmission optical microscope. The transmission optical microscope according to the present invention includes a light projector, a condenser lens, an objective lens, a light-receiving element, and a signal processor. The light projector emits light in a predetermined pattern and can be realized, for example, by a known projector. The condenser lens focuses light so that a focal plane is formed in an observation space where an object (sample) is placed. The objective lens focuses light that passes through this observation space. The light focused by the objective lens forms an image on the light-receiving surface of the light-receiving element. The light-receiving element converts the light focused by the objective lens into an electrical signal. The signal processor processes the electrical signal obtained from the light-receiving element to create an image. Here, the light projector emits striped light in multiple patterns in a three-dimensional observation space formed by an xyz Cartesian coordinate system, where the brightness is uniform in the y direction and periodically changes in the x direction on the focal plane (xy plane). The striped light is shifted in the x direction. It is particularly preferable that the striped pattern gradually shifts in the x direction over time. The signal processor then selectively acquires an image of an object at a desired depth in the z direction relative to the focal plane based on the electrical signals converted from the multiple light patterns. By controlling the light emission pattern and processing the signals in this way, a transmission optical microscope with tomographic observation capabilities can be realized.

[0009] In the transmission optical microscope according to the present invention, the signal processing unit preferably reduces the contrast of the image of an object that is out of the focal plane of the condenser lens in the z direction, thereby making it easier to distinguish between an object that is close to the focal plane of the condenser lens and an object that is out of the focal plane.

[0010] In the transmission optical microscope according to the present invention, the signal processing unit preferably reduces the contrast of the image of the object as the position in the z direction in the observation space becomes farther from the focal plane of the condenser lens, thereby facilitating observation of objects close to the focal plane of the condenser lens, and the image of an object away from the focal plane approaches the brightness of the background light and does not interfere with observation of the target object.

[0011] In the transmission optical microscope according to the present invention, the signal processing unit determines the brightness fluctuation range of the object image for each of the multiple patterns based on the electrical signals converted from the multiple patterns of light, and the smaller the fluctuation range, the more the contrast of the object image is reduced. When a striped pattern of light is irradiated onto an object while being shifted in the x direction, the brightness fluctuation range of the object image increases toward the focal plane of the light, and decreases toward the farther from the focal plane. Utilizing this principle, the signal processing unit processes the electrical signals using an algorithm that reduces the contrast of images with small brightness fluctuation ranges to generate an image.

[0012] In the transmission optical microscope according to the present invention, the number of patterns is preferably three or more. If the number of patterns is three or more, the tomographic observation function can be more effectively realized.

[0013] In the transmission optical microscope according to the present invention, it is preferable that the plurality of patterns are three types, A, B, and C, and the positions of the stripes for each pattern are shifted by 1 / 3 period in the x direction. In this case, the intensity distributions on the focal plane of the patterns A, B, and C are respectively expressed as follows: I A (x,y)=I0(1+cosβx), I B (x,y)=I0(1+cosβx+2π / 3), I C (x,y)=I0(1+cosβx+4π / 3) Also, let A, B, and C be the images S A (x,y), S B (x,y), S C (x,y), and the average value of the images A, B, and C is S=(S A +S B +S C ) / 3. In this case, the image of the object for each of the patterns A, B, and C can be calculated using the following calculation process: σ(x,y)=√([(S A -S) 2 +(S B -S)2 +(S C -S) 2 ] / 3)

[0014] The transmission optical microscope according to the present invention may further include a polarizer disposed between the light projecting unit and the observation space, and an analyzer disposed between the observation space and the light receiving element. In this manner, a pair of linear polarizers in a crossed Nicol state, i.e., a polarizer and an analyzer, is provided. The polarizer is placed before the light enters the observation object, and the analyzer is placed after the light has passed through the observation object. By applying the illumination method and tomographic observation method described above, a transmission optical microscope capable of both polarization observation and tomographic observation can be provided.

[0015] The transmission optical microscope according to the present invention may further include a first circular polarizing filter disposed between the light projecting unit and the observation space, and a second circular polarizing filter disposed between the observation space and the light receiving element. In this manner, a pair of circular polarizing filters in an extinction state is employed, with one filter, for example, a left-handed optical rotation filter, being placed before the light enters the observation object, and the other filter being placed after the light has passed through. By applying the illumination method and tomographic observation method described above, the transmission optical microscope can obtain observation images that are independent of the direction of birefringence.

[0016] The transmission optical microscope according to the present invention may further include a first polarization splitting / combining prism disposed between the light projecting unit and the observation space, and a second polarization splitting / combining prism disposed between the observation space and the light receiving element. In this manner, one of the two polarization splitting / combining prisms (including a Nomarski prism) is disposed before the light enters the observation object, and the other is disposed after the light has passed through the observation object. By applying the illumination method and tomographic observation method described above, a transmission optical microscope can be provided that combines polarization observation and differential interference contrast functions in addition to tomographic observation functions. [Effects of the Invention]

[0017] According to the present invention, a transmission optical microscope having a tomographic observation function can be provided. [Brief explanation of the drawings]

[0018] [Figure 1] FIG. 1 shows the configuration of a transmission optical microscope according to one embodiment of the present invention. [Figure 2] Figure 2 shows a schematic diagram of the principle of the tomographic observation function using a transmission optical microscope. [Figure 3] Figure 3 shows an image taken using a transmitted light microscope. [Figure 4] Figure 4 shows images taken using a transmission optical microscope, showing the difference in effect between with and without tomographic processing. [Figure 5] FIG. 5 shows the configuration of a modified example of the transmission optical microscope according to the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0019] Hereinafter, embodiments of the present invention will be described with reference to the drawings. The present invention is not limited to the embodiments described below, and includes appropriate modifications of the embodiments described below within the scope obvious to those skilled in the art. The figures in this application show three-dimensional coordinate axes formed by an xyz Cartesian coordinate system. The x direction is the horizontal direction, the y direction is the vertical direction, and the z direction is the depth direction. The z direction is parallel to the optical axis L of the camera, and the xy plane is a plane perpendicular to this optical axis. The xy plane is also parallel to the focal plane of the light output by the projector.

[0020] Fig. 1 shows a transmission optical microscope 100 according to one embodiment of the present invention. As shown in Fig. 1, the transmission optical microscope 100 includes a projector 10 (light projection unit), an optical system 20, a condenser lens 30, an objective lens 40, a digital camera 50, and a computer 60 (signal processing unit). A light-transmitting sample S to be observed by the transmission optical microscope 100 is placed in the observation space between the condenser lens 30 and the objective lens 40.

[0021] The projector 10 is a device for emitting light in a predetermined pattern. The projector 10 is preferably a DLP (Digital Lighting Processing) projector. The DLP projector includes a light source 11, a DMD (Digital Micromirror Device) 12, and a projection lens 13. The light source 11 is preferably capable of emitting white light, and a known lighting device such as an LED (Light Emitting Diode) can be used. The DMD 12 is a chip integrating millions of tiny movable mirrors, and the intensity of reflected light can be modulated by individually controlling each mirror. Although not shown in FIG. 1 , optical elements used in known DLP projectors, such as a condenser lens, a color wheel, and a relay lens, are provided between the light source 11 and the DMD 12. The light from the light source 11 is condensed onto the color wheel by the condenser lens, and the color wheel separates the light into red, blue, and green colored light, which is then introduced into the DMD 12 via the relay lens. This allows a DLP projector to control the brightness of each pixel and project a color image. Light whose reflection angle has been changed by the DMD 12 is introduced into the projection lens 13. The projection lens 13 then emits this light toward the subsequent optical system 20. DLP projectors are suitable for the transmission optical microscope 100 according to the present invention because they can easily enhance the contrast ratio.

[0022] In the present invention, the DMD 12 of the projector 10 is controlled to emit striped light on the focal plane F (xy plane) of the condenser lens 30, as shown in FIG. 1, whose brightness is uniform in the y direction and changes periodically in the x direction. Specifically, this striped light has linear high-brightness portions 12a extending in the y direction and linear low-brightness portions 12b similarly extending in the y direction, which are alternately arranged in the x-axis direction. While a sufficient brightness difference between the high-brightness portions 12a and the low-brightness portions 12b is sufficient, it is preferable that the brightness of the high-brightness portions 12a be set to at least twice the brightness of the low-brightness portions 12b. Furthermore, it is preferable that the high-brightness portions 12a and the low-brightness portions 12b be formed by forming a sinusoidal intensity distribution (striped pattern) on the focal plane F. The number of high-brightness portions 12a is preferably three or more, and more preferably five or more. Furthermore, it is preferable that the width (width in the x direction) of the high-brightness region 12a and the width of the low-brightness region 12b between the high-brightness regions 12a are substantially equal. "Substantially equal" means that the width of the low-brightness region 12b is within a range of ±10% of the width of the high-brightness region 12a. As will be described in detail later, the projector 10 outputs such striped light in multiple patterns that are slightly shifted in the x direction (see Figure 2). The DMD 12 of the projector 10 is capable of repeating images at tens of frames per second. When the individual micromirrors that make up the DMD 12 are controlled pixel-by-pixel at timings several orders of magnitude faster than a tenth of a second, the human eye or a camera sensor perceives the image as a (time-averaged) halftone.

[0023] The optical system 20 is composed of optical elements for guiding the light emitted from the projector 10 to the condenser lens 30. In the example shown in FIG. 1, the optical system 20 includes a collimator lens 21, a mirror 22, and an adjusting lens 23. The collimator lens 21 converts the diffused light emitted from the projector 10 into parallel light. The mirror 22 reflects the light that has passed through the collimator lens 21 and adjusts the direction of travel of the light. The adjusting lens 23 adjusts (expands or reduces) the beam diameter of the light reflected by the mirror 22 in order to introduce the light into the condenser lens 30.

[0024] The condenser lens 30 condenses the light that has passed through the optical system 20 and forms an image on a transparent sample S to be observed. A known microscope lens can be used as the condenser lens 30. As shown in FIG. 1, the light that has passed through the condenser lens 30 forms an image on a focal plane F. The sample S to be observed can be placed on this focal plane F. In FIG. 1, the dotted line frame between the condenser lens 30 and the objective lens 40 indicates the observation space for placing the sample S.

[0025] The objective lens 40 again focuses the light that has passed through the sample S, and forms an image on the light receiving element 51 of the digital camera 50. As with the condenser lens 30, a known microscope lens can be used for the objective lens 40. When placing the objective lens 40, the condenser lens 30 and the objective lens 40 are aligned so that the front focal point of the condenser lens 30 and the rear focal point of the objective lens 40 coincide with each other.

[0026] The digital camera 50 has a light receiving element 51 that receives light collected by the objective lens 40. The light receiving element 51 converts the received light into an electrical signal. A known photoelectric conversion element such as a CCD image sensor unit may be used as the light receiving element 51. The digital camera 50 is connected to a computer 60 by wire or wirelessly, and transmits the electrical signal obtained by the light receiving element 51 to the computer 60. The digital camera 50 may also include a photographing lens, a mechanical shutter, a shutter driver, a digital signal processor (DSP) that reads the amount of charge from the light receiving element 51 and generates image data, an IC memory, and the like. A configuration equivalent to this DSP may be provided in the computer 60, as described below.

[0027] The computer 60 processes the electrical signal obtained by the light-receiving element 51 as digital information based on a predetermined algorithm to generate an image. A known personal computer may be used as the computer 60. The computer 60 basically includes a processing unit, a storage unit, and a display unit. The processing unit may be a processor such as a CPU (Central Processing Unit) or a GPU (Graphics Processing Unit). The processing unit reads a program stored in the storage unit, expands it into main memory, and executes predetermined arithmetic processing according to the algorithm described in the program. The processing unit can also write and read the results of the calculations performed according to the program to and from the storage unit as appropriate. The storage unit is a component for storing information used in arithmetic processing by the processing unit and the results of the calculations. The storage function of the storage unit can be realized by nonvolatile memory such as an HDD or SSD. The storage unit may also function as a main memory for writing and reading intermediate data of arithmetic processing by the processing unit. The memory function of the storage unit can be realized by volatile memory such as RAM or DRAM. The display unit is a display device for displaying images generated by the processing unit. The display unit may be configured as a liquid crystal display or an organic EL display.

[0028] The computer 60 is also connected to the projector 10 and controls the light emitted by the projector 10. Specifically, the projector 10 adjusts the light intensity of the light source 11 and the reflection intensity of the tiny movable mirrors that make up the DMD 12 on a pixel-by-pixel basis based on control commands from the computer 60.

[0029] Next, with reference to FIG. 2, the principle of achieving a tomographic observation function using the transmission optical microscope 100 will be described. As shown in FIG. 1, light emitted from the projector 10 forms an image on the focal plane F (xy plane) of the condenser lens 30. FIG. 2 shows a striped light pattern in the xy plane projected onto this focal plane F. As mentioned above, the brightness of the striped light is uniform in the y direction and changes periodically in the x direction. As shown in FIG. 2, it is preferable to prepare at least three striped light patterns, A, B, and C. The light patterns are switched over over time in the order A → B → C → A → B → C... The striped patterns of the patterns A to B are all identical. However, compared to pattern A, pattern B is shifted by 1 / 3 of the period in the x direction. Furthermore, compared to pattern B, pattern C is shifted in the same x direction by 1 / 3 of the period. The period is the period at which pairs of high-brightness and low-brightness stripes 12a and 12b repeat. Therefore, if the distance in the x direction from the center of one high-brightness stripe 12a to the center of the next high-brightness stripe 12a is d, then 1 / 3 of the period is equal to d / 3.

[0030] Figure 2 also shows an xz cross section centered on the focal plane F. As shown in the xz cross section, when striped light is focused, areas of high brightness and areas of low brightness appear alternately in the x direction at the focal plane F, resulting in a large brightness difference. On the other hand, at positions shifted from the focal plane F in the z direction, the brightness difference in the x direction gradually decreases as the distance from the focal plane F increases.

[0031] As shown in FIG. 2, the sample S to be observed includes a circular object O1 and a diamond-shaped object O2. In this case, object O1 is located on focal plane F when viewed in the depth direction (z direction), while object O2 is located at a position offset from focal plane F in the depth direction. With the relative positions of objects O1 and O2 fixed, the striped light pattern is changed from A to B to C. Then, as shown in the xz cross-sectional view, the light intensity received by object O1 located on focal plane F fluctuates significantly with the change in the light pattern. Meanwhile, the light intensity received by object O2 located at a position offset from focal plane F in the z direction fluctuates less than that of object O1, even when the light pattern is changed. Thus, the fluctuation range (sharpness) of the light intensity received by each object O1 and O2 differs depending on the distance from focal plane F in the z direction. In the present invention, the difference in the intensity fluctuation range depending on the distance from focal plane F in the z direction is utilized to realize a tomographic observation function using the transmission optical microscope 100. 3, the image of a circular object O1 on the focal plane F can be made to have a clear contrast, while the image of a diamond-shaped object O2 located outside the focal plane F can have a reduced contrast. This allows the image of the object O1 on the focal plane F to be selectively acquired.

[0032] Next, in one embodiment of the present invention, a simple mathematical formula is used to explain how light emitted from an object to be observed is collected on the image plane of the camera as an image, i.e., how defocusing occurs depending on the degree of defocus, leading to a decrease in contrast. Following an algorithm based on this formula, computer 60 can reduce the contrast of the image of the object that is out of focus.

[0033] An object of observation located near focal plane F scatters the light illuminated by condenser lens 30. When the scatterer is located midway between condenser lens 30 and focal plane F (referred to as "in front of focal plane F"), the scattered light forms a somewhat blurred image on the focal plane of objective lens 40 (i.e., light receiving element 51). Furthermore, the component of this scattered light that exceeds the acceptance angle of objective lens 40 is lost. In the following analysis, the component of the light scattered by the object that reaches camera 50 (particularly light receiving element 51) is represented as a Gaussian beam with a divergence angle (of a Gaussian beam) that matches the acceptance angle of objective lens 40, and the light point can be considered as a Gaussian wave with a beam waist corresponding to that divergence angle (at infinity).

[0034] Here, the illumination light creates a sinusoidal intensity distribution (stripe pattern) on the focal plane F. This intensity distribution is expressed as follows: [Formula 1] TIFF2025138250000002.tif9166Note that I(x,y) is the intensity at position (x,y). I0 is the intensity of the light source (maximum intensity). β is the spatial angular frequency of the interference fringes. γ is the phase of the fringes.

[0035] Furthermore, at a distance z away from the focal plane F, the illumination light is considered to have a distribution on the focal plane that is the convolution integral of a Gaussian cone. A remote infinitesimal Gaussian wave source with a radiation angle that matches the NA of the illumination system, uncorrelated, and a beam waist on the focal plane, and a half-angle of divergence θ, is considered to cover the focal plane according to the sinusoidal intensity distribution given by the above equation. Once it leaves the focal plane F, it spreads according to the propagation laws of a coherent Gaussian wave. At a distance z away from the focal plane F, the Gaussian waves are superimposed with power to determine the excitation intensity. The propagation of the illumination light is symmetric with respect to the focal plane F.

[0036] Here, the formula for convolution is expressed as follows: [Formula 2] TIFF2025138250000003.tif12166 where J A(x,y) is the intensity distribution at position (x,y). α is the initial phase difference. ξ is the integral variable. θ is the half angle of the divergence of the stripe illumination light. z is the distance from the focal plane in the optical axis direction. Doing this also gives the following formula: [Formula 3] TIFF2025138250000004.tif15166J A is the same type as I(x,y), but the fluctuation range (sharpness) of the light intensity received by the observed object as the light pattern fluctuates is multiplied by K (reduction, K<1, fault effect).

[0037] The movement of the stripe pattern for each 1 / 3 period is written as (A, B, C, A, B, C...) as in Figure 2. The illumination light (J A (x,y),J B (x,y),J C The object to be observed at a point (x0, y0, z) shifted in the z direction from the focal point F under illumination light of pattern A creates an image that spreads across the camera image plane, and the signal is expressed by the following equation: [Formula 4] TIFF2025138250000005.tif15166where, S A (x, y) is the intensity at the position (x, y) when illuminated with illumination light of pattern A. K is the contrast. φ is the half angle of acceptance of the objective lens. The same applies to the illumination light of patterns B and C. [Formula 5] TIFF2025138250000006.tif27166

[0038] Also, the mean of the image is S=(S A +S B +S C ) / 3. The degree of signal sharpness associated with fluctuations in the light pattern is defined by the following fluctuation width σ(x, y): [Formula 6] TIFF2025138250000007.tif9166When expanded, this becomes: [Formula 7] TIFF2025138250000008.tif14166

[0039] Therefore, we can say that σ(x,y) = constant × formula A × formula B. Formula A is as follows. [Formula 8] TIFF2025138250000009.tif14166 Thus, equation A is a function of x and y and describes the blurring effect caused by out-of-focus. And equation B is: [Formula 9] TIFF2025138250000010.tif10166Equation B represents the tomographic effect, which is the subject of this invention. Therefore, the computer simply reduces the contrast by a factor of B for the image of the object on the plane where the defocus is represented by z (the distance from the focal plane F). As the object moves away from the focal plane F, it approaches the uniform brightness of the background light and becomes invisible. Note that the light from point (x0, y0, z) is the overlap of light emitted by the object and background light. Because the background light is uniform, the analysis focuses only on the light diffraction of the light intensity obtained by subtracting the background light intensity from the light intensity emitted by the object, and what is actually observed is the uniform light intensity superimposed on this. This application employs such an analysis and display method.

[0040] The effects of the present invention will be explained with reference to Figures 1 and 2. First, striped illumination light is generated from a DLP type projector 10 having a light source 11 (LED) and a DMD 12 (a movable micromirror array) controlled by a computer 60. The light emitted from the projector 10 then passes through an optical system 20 including lenses 21, 23 and a mirror 22, is condensed by a condenser lens 30 on a focal plane F in the observation space, and is then focused by an objective lens 40 on a light receiving element 51 of a camera 50 to obtain an image. Note that the linearity between the power of incident light and the output electrical signal of each sensor in the camera 50 is corrected in advance. The computer 60 calculates the linearity of each pattern I of illumination light. A (x,y), I B (x,y), I C Array pattern J of electrical output corresponding to (x,y) A (x,y), JB (x,y), J C Extract the image σ(x,y) from (x,y).

[0041] As an example of the image of an object to be observed, when a circular object and a diamond-shaped object, whose object position is shown in Figure 2, are both initially located on the focal plane z = z1, the image of each object will be as shown in Figure 3(a). Next, when the circular object remains fixed at the focal plane z = z1 and only the diamond-shaped object is moved in the z direction from the focal plane (z = z1) by a certain length L, the images of each object will be as shown in Figure 3(b). If only the diamond-shaped object is further moved in the z direction by the same distance, the image will be as shown in Figure 3(c). This simply shows the effect of being out of focus. In Figure 3, the illumination light is of uniform brightness, just like a normal microscope.

[0042] In contrast, Figure 4 shows the effect of performing the signal processing described above using the multiple striped illumination patterns shown in Figure 2. When both the circular object and the diamond-shaped object are initially located on the focal plane z = z1, the images of each object are as shown in Figure 4(a). Next, while the circular object remains fixed at the focal plane z = z1, only the diamond-shaped object is moved a certain distance L in the z direction from the focal plane (z = z1). When the signal processing described above is performed, the images of each object are as shown in Figure 4(b). Note that this certain distance L is the value of z at which the aforementioned "amount B representing the tomographic effect" is halved (the amount of defocus at which the contrast is halved). The defocus φz in equation A is also determined corresponding to L or z. The image of the diamond-shaped object shown in Figure 4(b) has the same blur as Figure 3(b), but the light-dark contrast is lower than in Figure 3(b). If the diamond-shaped object is further moved the same distance in the z direction, the image becomes as shown in Figure 4(c). The image of the diamond-shaped object shown in Figure 4(b) is blurred more than that of Figure 3(b), but the loss of contrast is even more pronounced. As is clear from the mathematical explanation above, blur increases linearly with distance, but the loss of contrast is Gaussian. The contrast of the diamond image is so weak that it is difficult to see, and the tomographic effect is evident.

[0043] Next, a modified example in which another function is added to the transmission optical microscope 100 will be described with reference to FIG.

[0044] FIG. 5(a) shows an example of a transmission optical microscope 100 equipped with a polarized light observation function. Polarized light microscopes are used, for example, when birefringence exists in a transparent object being observed due to stress or other factors. In other words, polarized light microscopes are used to observe the effect of light being polarized in a certain state entering the object, causing the polarization state of light exiting the object to change from that of the incident light. The most common configuration is described below. As shown in FIG. 5(a), the birefringence effect can be observed by adding a pair of polarizers 71 and 72 in a crossed Nicol configuration to the front and rear of the observation space in addition to the basic configuration of the transmission optical microscope 100 shown in FIG. 1. Specifically, a polarizer 71 (polarizing plate) is placed between the optical system 20 (adjusting lens 23) and the focusing lens 30, and an analyzer 72 (polarizing plate) is placed between the objective lens 40 and the camera 50. In this configuration, sensitivity differs depending on the principal axis directions of the polarizers 71 and 72 and the birefringence direction of the object being observed. The sensitivity is maximized when the birefringence direction forms an angle of ±45 degrees with respect to the two orthogonal principal axis directions of the crossed Nicols, and is zero when the birefringence direction is zero or 90 degrees. This directional dependency is both an advantage and a disadvantage. It is necessary to measure the object of observation in two directions that differ by 45 degrees with respect to the principal axis of the polarizer 71, but more detailed information can be obtained. Even with the modified example shown in Figure 5(a), it is possible to perform tomographic observation by performing predetermined calculations on an image in which the illumination light has three different stripe patterns, as shown in Figure 2, etc.

[0045] Another method is shown in Figure 5(b). In the example shown in Figure 5(b), in addition to the basic configuration of the transmission optical microscope 100 shown in Figure 1, a pair of circular polarizing filters 73, 74 that are mutually extinct are inserted before and after the observation space. That is, a first circular polarizing filter 73 is placed between the optical system 20 (adjusting lens 23) and the condenser lens 30, and a second circular polarizing filter 74 is placed between the objective lens 40 and the camera 50. Note that these circular polarizing filters 73, 74 may be realized, for example, by combining a linear polarizer and a quarter-wave plate. For example, the first circular polarizing filter 73 linearly polarizes unpolarized illumination light using a linear polarizer, then circularly polarizes it using a quarter-wave plate, and irradiates this circularly polarized light onto the observation object. In addition, the light that passes through the observation object is introduced to the second circular polarizing filter 74. The light introduced to the second circular polarizing filter 74 passes through the quarter-wave plate and is then guided to the linear polarizer. When an observation object without birefringence is inserted, the angular relationship between the two linear polarizers of each circular polarizing filter 73, 74 is maintained so that an overall extinction state can be achieved. Note that even in the modified example shown in Fig. 5(b), it is possible to perform tomographic observation by performing predetermined calculation processing on an image in which the illumination light has three different stripe patterns, as shown in Fig. 2 etc.

[0046] In the modified examples shown in Figures 5(a) and 5(b), the signal light intensity may be too low because only birefringence in a specific portion of the optical path is observed. To avoid this problem, the period of the stripe pattern of the irradiated light can be increased as needed. This increases the length of the optical path used for tomography and strengthens the signal light. However, there is a trade-off between resolution and brightness.

[0047] Next, Figure 5(c) shows an example of a transmission optical microscope 100 to which a differential interference function has been added. A differential interference microscope uses a first linear polarization separation prism to separate and propagate incident light (linearly polarized light), propagating it as light slightly shifted at the focal plane where the object of observation is located, and then combines it into a single light beam using a second linear polarization combination prism. If there is an optical path difference between the two parallel light beams, this optical path difference is observed as a change in the polarization state of the combined light. A differential interference microscope is suitable for observing the inside of a transparent object because it can convert the distribution of the spatial optical path difference (or phase difference) of the object of observation into a spatial distribution of the polarization state of light for observation.

[0048] 5(c), the order of the optical path of light is as follows: optical system 20 (adjusting lens 23), first polarizer 71, first differential interference contrast (DIC) prism 75, condenser lens 30, observation space where the sample is placed, objective lens 40, second polarizer 76, second differential interference contrast (DIC) prism 77, and analyzer 72. To provide such a differential interference microscope with a tomographic observation function, as described above, illumination light having three different striped intensity distributions is irradiated onto the sample, and calculations are performed on the observation images obtained for each.

[0049] In the above description of the present invention, the embodiments of the present invention have been described with reference to the drawings in order to express the contents of the present invention. However, the present invention is not limited to the above embodiments, and includes modifications and improvements that are obvious to those skilled in the art based on the matters described in the present specification. [Explanation of symbols]

[0050] 13...Projection lens 20...Optical system 21...Collimating lens 22...Mirror 23...Adjusting lens 30...Condenser lens 40...Objective lens 50...Camera 51...Photodetector 60...Computer 71...Polarizer 72...Analyzer 73...Circular polarizing filter 74...Circular polarizing filter 75...Prism 76...Polarizer 77...Prism 100...Transmission optical microscope S...sample O...object F…focal plane L…optical axis

Claims

1. a light projection unit that emits light of a predetermined pattern; a condenser lens that condenses the light so that a focal plane is formed in an observation space in which an object is placed; an objective lens that condenses the light that has passed through the observation space; a light receiving element that converts the light collected by the objective lens into an electrical signal; a signal processing unit that processes the electrical signal to generate an image; the light projecting unit emits striped light, the brightness of which is uniform in the y direction and which changes periodically in the x direction, on the focal plane in a three-dimensional observation space formed by an xyz Cartesian coordinate system, in a plurality of patterns by shifting the position of the striped pattern in the x direction; The signal processing unit selectively acquires an image of an object at a desired depth in the z direction relative to the focal plane based on the electrical signals converted from the plurality of patterns of light. Transmitted light microscope.

2. The signal processing unit reduces the contrast of an image of an object that is deviated from the focal plane of the focusing lens in the z direction.

2. The transmission optical microscope according to claim 1.

3. The signal processing unit reduces the contrast of the image of the object as the position in the z direction in the observation space becomes farther from the focal plane of the condenser lens.

2. The transmission optical microscope according to claim 1.

4. The multiple patterns are three or more.

2. The transmission optical microscope according to claim 1.

5. The plurality of patterns are three types, A, B, and C, and the positions of the stripes are shifted by 1 / 3 of a period in the x direction for each pattern, The intensity distributions of the patterns A, B, and C on the focal plane are respectively expressed as I A (x,y)=I 0 (1+cosβx)、 I B (x,y)=I 0 (1+cosβx+2π / 3)、 I C (x,y)=I 0 (1+cosβx+4π / 3), Images A, B, and C, respectively, S A (x,y)、 S B (x,y)、 S C Define (x,y), The average of images A, B, and C is S=(S A +S B +S C ) / 3, The image of the object for each pattern A, B, and C is σ(x,y)=√([(S A -S) 2 +(S B -S) 2 +(S C -S) 2 ] / 3) This is calculated by the following calculation:

2. The transmission optical microscope according to claim 1.

6. a polarizer disposed between the light projecting unit and the observation space; an analyzer disposed between the observation space and the light receiving element; 2. The transmission optical microscope according to claim 1.

7. a first circular polarizing filter disposed between the light projecting unit and the observation space; a second circular polarizing filter disposed between the observation space and the light receiving element; 2. The transmission optical microscope according to claim 1.

8. a first polarization separation / combination prism disposed between the light projection unit and the observation space; a second polarization separation / combination prism disposed between the observation space and the light receiving element; 2. The transmission optical microscope according to claim 1.

Images