JP2008242022A - Polarizing microscopic observation method and polarizing microscope system - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To achieve highly spatial differentiation of not only a transparent crystal-like substance but also a living specimen and to achieve observation of a dynamic change in such specimens at increased time resolution without dyeing them. <P>SOLUTION: The polarizing microscopic observation method using a polarizing microscope is disclosed in which a polarizer 6 is disposed in a specimen illuminating optical path and an analyzer 7 is disposed in a crossed Nichol state in an optical path from an objective lens 2 to an imaging face 3. The method includes: disposing a variable phaser 9 between the polarizer 6 and a specimen S or between the specimen S and the analyzer 7; converting a phase difference given by the variable phaser member 9 into three differences different from one another in terms of a lapse of time; imaging a polarizing microscopic image on a sheet each time a phase difference is given; and calculating the phase difference distribution image of the specimen S from the three captured images. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a polarizing microscope observation method and a polarizing microscope system, and in particular, in an optical microscope using linearly polarized light as irradiation light, a voltage is applied to a variable phase shifter over time, and a polarizing microscope image is synchronized with the voltage application. The present invention relates to a method and system for obtaining a phase difference distribution image of a sample by acquiring and processing acquired image information.

  In a conventional transmission-type ordinary optical microscope, a transparent biological sample or a transparent crystal / amorphous substance has a low contrast and cannot be identified. In the case of living organisms, a method of observing by staining with a dye has been taken.

  In addition, even in a fluorescence microscope known for high sensitivity, in the case of biological samples that do not emit intrinsic fluorescence, it is necessary to use them after staining with fluorescent dyes. It is difficult to ignore.

  In addition, the polarization microscope that has been used in the past can observe even ultra-thin sections of transparent fibrous materials, biological samples, rocks, and crystals by utilizing the difference in birefringence using linearly polarized light. . However, there are drawbacks in that the amount of retardation (phase difference) is not quantified, observation takes time, and skill and experience are required for handling the polarizing microscope. Further, it is also inconvenient when an environmental condition whose structure is dynamically changed is given or an object that changes like a living thing.

  As a method for improving this, a polarizing microscope using liquid crystal control by Oldenburg (1996) of the United States was invented (Patent Document 1) and made to overcome these drawbacks.

However, unlike the method of the present invention which will be described later, since the voltage is applied to the two liquid crystal plates, the system becomes complicated. Further, since the change of the liquid crystal polymer with respect to the voltage takes several tens of milliseconds, it is essentially impossible to acquire images at high speed. In addition, it is necessary to perform image processing on four screens to form one screen, and when a moving image is created, one screen is synthesized with four screens, so that there is a disadvantage that time resolution is low.
US Pat. No. 5,521,705

  The present invention has been made in view of the above-described problems of the prior art, and its purpose is not only a transparent crystalline substance but also a living biological sample with high spatial discrimination ability and time resolution. The dynamic change of such a sample can be observed without staining.

The polarizing microscope observation method of the present invention uses a polarizing microscope in which a polarizer is disposed in a single-wavelength sample illumination optical path, and an analyzer is disposed in a crossed Nicol state in the optical path from the objective lens to the imaging surface. In the observation method,
A variable phase shifter is arranged between the polarizer and the sample or between the sample and the analyzer, and the phase difference given by the variable phase shifter is set to three phase differences different from each other with time. In this method, a single polarizing microscope image is taken and a phase difference distribution image of the sample is calculated from the three acquired images.

In this case, the phase differences given by the variable phase shifter are θ ₁ , θ ₂ , and θ ₃ , respectively, and the light intensity on the imaging surface when the phase differences θ ₁ , θ ₂ , and θ _{3 are} given is I (x) _{When 1} , I (x) ₂ , and I (x) ₃ (x is a coordinate on the imaging surface), it is desirable to calculate the phase difference distribution image R (x) by performing the calculation of Expression (29). .

R (x) = 1/2 ×
| {(Θ ₂ ² −θ ₁ ² ) −p (θ ₃ ² −θ ₁ ² )}
÷ {(θ ₂ −θ ₁ ) −p (θ ₃ −θ ₁ )} | (29)
However,
p = (I (x) ₂ −I (x) ₁ ) / (I (x) ₃ −I (x) ₁ ) (24)
It is.

  Further, it is desirable that the variable phase shifter is composed of a Pockels cell or a liquid crystal element.

  Further, it is desirable that the phase difference given by the variable phase shifter is 1/36 or less of the wavelength.

Further, it is desirable that the phase difference given by the variable phase shifter is θ ₁ = 0, θ ₂ = a, θ ₃ = −a.

  Further, the three different phase differences are periodically given, and the consecutive images captured at the respective phase differences are respectively calculated from the three consecutive images obtained by shifting the photographing time intervals to obtain the phase difference distribution image. Can be calculated continuously for each photographing time interval.

The polarization microscope system of the present invention uses a polarization microscope in which a polarizer is arranged in a single-wavelength sample illumination optical path, and an analyzer is arranged in a crossed Nicols state in the optical path from the objective lens to the imaging surface. In the microscope system,
A variable phase shifter is disposed between the polarizer and the sample or between the sample and the analyzer;
Control means for causing the variable phase shifter to generate three phase differences different from each other over time; imaging means for capturing one polarization microscope image in synchronization with the variable phase shifter when each phase difference is applied; The image processing apparatus includes a calculation unit that calculates a phase difference distribution image of the sample from the three captured polarization microscope images, and a display unit that displays the calculated phase difference distribution image of the sample.

  In this case, the control means controls the three phase differences different from each other by the variable phase shifter to periodically change, and the imaging means synchronizes with each phase difference of the variable phase shifter to each one sheet. By taking a polarization microscope image and calculating one phase difference distribution image of the sample from three consecutive polarization microscope images while shifting the time interval by which the control means changes the phase difference by the control means, the imaging means It is also possible to obtain phase difference distribution images successively in order at the same time intervals as in the case of shooting in FIG.

  According to the polarization microscope observation method and polarization microscope system of the present invention, if there is a structure such as a birefringent molecule or substance in a dark screen, the magnitude of the phase difference due to the structure can be detected as the light intensity. In addition, it is possible to measure the amount of substances quantitatively.

  In addition, it is possible to discriminate with a very high spatial discriminating ability that a molecule having a size of several tens of nm is observed as an image that shines in the dark.

  In addition, it is possible to collect very high-sensitivity image data without staining a transparent substance, and observation is possible even when an organism is affected by staining.

  Further, by collecting information in the depth direction by using a high-magnification objective lens and reducing the depth of focus, it is possible to construct a three-dimensional solid such as a cell.

  In addition, it is possible to observe the cells in the living tissue in an unstained state and the internal structure of the cells dynamically and with a high spatial discrimination ability.

  Hereinafter, the principles and embodiments of the polarizing microscope observation method and the polarizing microscope system of the present invention will be described with reference to the drawings.

  FIG. 1 is a diagram showing a configuration of one embodiment of a polarizing microscope system of the present invention. The polarizing microscope includes a stage 1 on which a sample S is placed, an objective lens 2, and a sample S enlarged by the objective lens 2. Of the CCD camera 3, the light source 4, the condenser 5, the linear polarizer (polarizer, polarizer) 6 disposed between the light source 4 and the condenser 5, the objective lens 2, and the CCD camera 3. A linear polarizer (analyzer, analyzer) 7 disposed between them, an interference filter 8 disposed between the light source 4 and the linear polarizer (polarizer) 6 and transmitting illumination light of a single wavelength, and the objective lens 2 A Pockels cell 9 is arranged between linear polarizers (analyzers) 7 and generates a predetermined phase difference. An image photographed by the CCD camera 3 is taken into a computer 10, and a Pockels cell is used. Phase difference of 9 is controlled by signals from the computer 10 is input to the Pockels cell 9 by the Pockels cell controller 12 via the parallel port 11. Further, the phase difference distribution image calculated by the computer 10 is displayed on the monitor 13.

  In the polarization microscope system according to the present invention, since the phase is important information, light of a single wavelength is used as an irradiation light source. Therefore, the light source 4 is a laser light source having a single wavelength or a light source having a wide wavelength. In the case of a certain mercury lamp, xenon lamp, metal halide lamp, halogen lamp, etc., light having a single wavelength that has passed through the interference filter 8 is used. When a laser light source is used as the light source 4, the interference filter 8 may be omitted.

  The linear polarizer (polarizer) 6 and the linear polarizer (analyzer) 7 are arranged in a crossed Nicols state in which the transmission axes are orthogonal to each other. The optical axis of the Pockels cell 9 is the transmission axis of the polarizer 6 and the analyzer 7. It arrange | positions so that the angle of 45 degrees may be made with respect to each transmission axis.

  The same effect can be obtained even if the Pockels cell 9 is disposed between the linear polarizer 6 and the sample S.

  In such an optical system, in the state where the sample S is not present, the optical microscope is arranged so that the screen imaged by the CCD camera 3 is dark, and the extinction ratio (extinction coefficient = incident light luminance / emitted light luminance, extinction). The larger the factor) is, the darker the entire field of view of the microscope is. Therefore, based on the birefringent substance of the sample S to be observed, it becomes possible to pass through the analyzer 7 according to the amount of phase change (phase difference). Depending on the magnitude of the amount of phase change, the sample S is observed with a strong glow in the field of view, so that a large detection sensitivity can be obtained.

  By applying voltage to the Pockels cell 9 or a liquid crystal plate composed of a liquid crystal element used in place of it, or a variable phase shifter (compensator) based on other electro-optic effects, a phase difference is produced between two linearly polarized beams whose vibration planes are orthogonal to each other. Give rise to This phase difference is controlled by applying a voltage from the computer 10 to the Pockels cell 9 via the parallel port 11 and the Pockels cell controller 12. The voltage applied at this time is a voltage that produces a phase difference of about 1/36 of the wavelength in linearly polarized light (about 15 nm when the wavelength used is 540 nm of green light) or less.

  An image obtained in synchronism with the control voltage is acquired from the CCD camera 3 under the control of the computer 10, and the following arithmetic processing is performed for each pixel to display an image on the monitor 13.

  It is also possible to manually use a variable phase shifter (compensator). As an example of the variable phase shifter, a peeled mica plate can be used.

  Hereinafter, an arithmetic algorithm for image processing will be described.

Let I _n (x) be the luminance at the pixel at address x of the nth image of the CCD camera 3 that has passed through the analyzer 7. The phase difference given by the Pockels cell 9 is assumed to be θ _n . The phase difference (retardation) due to the birefringence of the sample S at the pixel x is defined as θ ₀ (x). The light intensity I ₀ (x) incident on the analyzer 7 which is the final optical element is set, and the minimum value of I _n (x) (light intensity in the crossed Nicols state) is set to I _m (x). I _n (x) is a value obtained by actual observation, and θ _n is a value given by the observer. Now, in the CCD camera 3, focusing on the x-th address of the nth image, the retardation θ ₀ (x) is obtained from the formula relating to luminance. In the following description, x is omitted.

I _n = I ₀ · sin ² (θ _n −θ ₀ ) + I _m (1)
The first item of the equation (1) is the amount of light passing through the analyzer 7 based on the deviation (θ _n −θ ₀ ) from the phase difference (retardation) θ _{0 in} the crossed Nicols state.

Assuming that θ ₀ and θ _n are also very small angles, Equation (1) is approximately
I _n = I ₀ · (θ _n −θ ₀ ) ² + I _m (2)
It becomes.

Here, from the three equations relating to the light intensities I ₁ , I ₂ , and I ₃ obtained when the phase differences θ ₁ , θ ₂ , and θ ₃ caused by the three kinds of voltages applied to the Pockels cell 9 are given, this ternary is obtained. By solving the simultaneous equations, the unknowns I ₀ , I _m , and θ ₀ are generally obtained. A phase difference (retardation) θ ₀ distribution image is obtained by displaying θ ₀ at a location for each pixel. By displaying θ ₀ that changes every moment, a retardation distribution change moving image of the sample S can be obtained.

As an example, the light intensity recorded by the CCD camera 3 when a phase difference of θ ₁ = 0, θ ₂ = a, and θ ₃ = −a is given by three kinds of voltages applied to the Pockels cell 9 now. Assuming I ₁ , I ₂ , and I ₃ (the luminance of one pixel at the same address in three images), the phase difference (retardation) θ ₀ due to the birefringence of the sample S is expressed as the following equation as a solution of the ternary simultaneous equations: It is obtained by

Substituting θ ₁ = 0, θ ₂ = a, and θ ₃ = −a into equation (2) to solve the simultaneous equations.

I ₁ = I ₀ · θ ₀ ² + I _m (3)
I ₂ = I ₀ · (a ² -2aθ ₀ + θ ₀ ² ) + I _m (4)
I ₃ = I ₀ · (a ² + 2aθ ₀ + θ ₀ ² ) + I _m (5)
From equation (3)
I _m = I ₁ −I ₀ · θ ₀ ² (6)
Substituting equation (6) into equations (4) and (5),
I ₂ = I ₀ a ² -2 I ₀ aθ ₀ + I ₁ (7)
I ₃ = I ₀ a ² + 2I ₀ aθ ₀ + I ₁ (8)
From equations (7) and (8),
I ₀ a ² + I ₁ = I ₂ +2 I ₀ aθ ₀ (9)
I ₀ a ² + I ₁ = I ₃ -2 I ₀ aθ ₀ (10)
From equation (9) = equation (10)
I ₂ + 2I ₀ aθ ₀ = I ₃ −2I ₀ aθ ₀ (11)
From equation (11)
4I ₀ aθ ₀ = I ₃ −I ₂ (12)
I ₀ = (I ₃ −I ₂ ) / 4aθ ₀ (13)
Substituting equation (13) into equation (6),
I _m = I ₁ − (I ₃ −I ₂ ) θ ₀ / 4a (14)
Substituting Equations (13) and (14) into Equation (5),
I ₃ = {(I ₃ −I ₂ ) / 4aθ ₀ } (a ² + 2aθ ₀ + θ ₀ ² )
+ I ₁ − (I ₃ −I ₂ ) θ ₀ / 4a (15)
4aθ ₀ I ₃ = (I ₃ −I ₂ ) (a ² + θ ₀ ) ²
+ 4aθ ₀ I ₁ − (I ₃ −I ₂ ) θ ₀ ²
4aθ ₀ I ₃ = (I ₃ −I ₂ ) (a + 2θ ₀ ) a + 4aθ ₀ I ₁
4θ ₀ I ₃ = (I ₃ −I ₂ ) (a + 2θ ₀ ) + 4θ ₀ I ₁
θ ₀ (4I ₃ −2I ₃ + 2I ₂ −4I ₁ ) = (I ₃ −I ₂ ) a
θ ₀ = (I ₃ −I ₂ ) a / (4I ₃ −2I ₃ + 2I ₂ −4I ₁ )
= (I ₃ -I ₂ ) a / (2I ₃ + 2I ₂ -4I ₁ ) (16)
However, I ₁ , I ₂ , and I ₃ represent the luminance of one pixel at the same address in the acquired three images. a is a constant given by the observer.

Therefore, the retardation value R (x) of the sample S is
R (x) = | θ ₀ (x) |
= | (I ₃ (x) −I ₂ (x)) a
÷ {2I ₃ (x) + 2I ₂ (x) -4I ₁ (x)} | (17)
It becomes.

The above is a case where θ _n (phase difference given by the Pockels cell 9) in the formula (2) is set to three specific values: 0, a, and −a. As described above, the unknown is I ₀ , Since I _m and θ ₀ are three, in principle, three different values are given to θ _n in the equation (2), and an image obtained by the CCD camera 3 is fetched into the computer 10 in synchronization therewith. A retardation distribution image R (x) of the sample S can be obtained by performing the same arithmetic processing for each pixel and displaying an image on the monitor 13.

A general solution regarding the case where the phase differences θ ₁ , θ ₂ , θ ₃ of the phase differences given by the Pockels cell 9 are set as follows is shown below.

Substituting θ ₁ , θ ₂ , and θ ₃ into θ _n in Equation (2), the simultaneous equations are solved.

I ₁ = I ₀ · (θ ₁ −θ ₀ ) ² + I _m (18)
I ₂ = I ₀ · (θ ₂ −θ ₀ ) ² + I _m (19)
I ₃ = I ₀ · (θ ₃ −θ ₀ ) ² + I _m (20)
Subtracting equation (18) from equations (19) and (20) respectively,
I ₂ −I ₁ = I ₀ {(θ ₂ −θ ₀ ) ² − (θ ₁ −θ ₀ ) ² }
= I ₀ (θ ₂ −θ ₁ ) (θ ₂ + θ ₁ −2θ ₀ ) (21)
I ₃ −I ₁ = I ₀ {(θ ₃ −θ ₀ ) ² − (θ ₁ −θ ₀ ) ² }
= I ₀ (θ ₃ −θ ₁ ) (θ ₃ + θ ₁ −2θ ₀ ) (22)
From the above formula (21) and formula (22),
(I ₂ -I ₁ ) / (I ₃ -I ₁ )
= {(Θ ₂ −θ ₁ ) (θ ₂ + θ ₁ −2θ ₀ )}
÷ {(θ ₃ −θ ₁ ) (θ ₃ + θ ₁ −2θ ₀ )} (23)
Now
p = (I ₂ −I ₁ ) / (I ₃ −I ₁ ) (24)
And put
p (θ ₃ −θ ₁ ) (θ ₃ + θ ₁ −2θ ₀ )
= (Θ ₂ −θ ₁ ) (θ ₂ + θ ₁ −2θ ₀ ) (25)
p = (I ₂ −I ₁ ) / (I ₃ −I ₁ ) ≠ (θ ₂ −θ ₁ ) / (θ ₃ −θ ₁ )
... (26)
When
θ ₀ = 1/2 × {(θ ₂ ² −θ ₁ ² ) −p (θ ₃ ² −θ ₁ ² )}
÷ {(θ ₂ −θ ₁ ) −p (θ ₃ −θ ₁ )} (27)
It can be solved. Therefore, the retardation value R (x) of the sample S when arbitrary phase differences θ ₁ , θ ₂ , θ ₃ are given by the Pockels cell 9 is
R (x) = | θ ₀ (x) | = 1/2 × | {(θ ₂ ² −θ ₁ ² ) −p (θ ₃ ² −θ ₁ ² )}
÷ {(θ ₂ −θ ₁ ) −p (θ ₃ −θ ₁ )} | (29)
It becomes. However, p is Formula (24):
p = (I (x) ₂ −I (x) ₁ ) / (I (x) ₃ −I (x) ₁ ) (24)
Given in.

  As described above, in the polarizing microscope system of the present invention, a single liquid crystal plate can be used instead of the two liquid crystal plates used in the polarizing microscope according to Patent Document 1. Alternatively, when the Pockels cell 9 using the Pockels effect of changing the phase difference (retardation) of birefringence by applying a voltage to the Pockels element is used as a phase shifter, the driving speed is 10 microseconds or less, and the liquid crystal The retardation distribution image can be observed at a speed 1000 times higher than that of the above.

  In the polarization microscope system of the present invention, since it is possible with only one liquid crystal plate or one Pockels cell, the driving electric circuit and computer control are simplified.

  Furthermore, since only three screens are required for the construction of one image, three screens are required compared to the case where four screens are required in the polarizing microscope according to Patent Document 1, and image display can be performed in a shorter time. It becomes. In the present invention, the fundamental improvements as described above are made.

In addition, it is possible to realize shooting of an image in which three phase amounts are changed at regular time intervals (an image in which the phase difference is sequentially changed from θ ₁ → θ ₂ → θ _{3 in the} Pockels cell 9). In the following, the phase difference given by the Pockels cell 9 continuously and periodically is changed to three stages, for example, + a, 0, -a, and the time-varying retardation from the image obtained by the CCD camera 3 based on the equation (17). The process of obtaining the distribution images in order is schematically shown in FIG. In FIG. 2, the time axis is taken in the right direction. FIG. 2A shows a voltage waveform that can be applied to the Pockels cell 9. As shown in FIG. 2A, a voltage that changes stepwise from + V → 0 → −V at a time interval Δt is applied to the Pockels cell 9 at a period T. Then, the phase difference generated in the Pockels cell 9 becomes a phase difference that changes in the cycle T from + a → 0 → −a at the time interval Δt, as shown in FIG. Then, as shown in FIG. 2C, the images taken by the CCD camera 3 at the respective phase differences are the above-described phase differences + a, 0, -a, + a, 0, -a, + a, 0. .., −a,... Corresponding to changes in the order of the images with the signs +, 0, −, +, 0, −, +, 0, −,. can get. Then, from the above principle, from the three consecutive images obtained by shifting by the time interval Δt, as shown in FIG. 2 (d), one retardation distribution image (1) → (2) → (3 ) → (4) → (5) → (6) → (7) → (8) → (9)... Are sequentially obtained at the time interval Δt. For this reason, the retardation distribution image can be obtained successively in order at the same time interval as the photographing by the CCD camera 3, and a moving image can be created.

  In this regard, in the method of Patent Document 1, as shown in FIG. 2 (e), one set of four images (one set of three images in FIG. As is apparent from this figure, the time resolution four times that of the method disclosed in Patent Document 1 is obtained.

  As is clear from the above description, in the polarizing microscope system of the present invention, when there is a structure such as a birefringent molecule or substance in a dark screen, the magnitude of the phase difference due to the structure is the light intensity. Since it can be detected, it is possible to measure the amount of substance quantitatively. This is a significant feature that even transparent materials that cannot be confirmed with a conventional microscope can be observed if their birefringence differs from the surrounding materials.

  In addition, it is possible to discriminate with a very high spatial discrimination ability that a molecule of several tens of nanometers is observed as an image that shines in the dark. This is a very high spatial discrimination ability compared with the spatial resolution that the separation of a distance of two hundred and several tens of nm is theoretically possible as an optical microscope.

  Further, it is important in that it is possible to collect highly sensitive image data without staining a transparent substance, and observation is possible even when the influence of staining occurs in living organisms.

  Further, by collecting information in the depth direction by using a high-magnification objective lens and reducing the depth of focus, it is possible to construct a three-dimensional solid such as a cell.

  From the above, it has the characteristic that it can observe dynamically about the cell in the biological tissue in the living state without dyeing | staining, and the internal structure of a cell. That is, it is possible to discriminate with a high spatial discrimination ability a fibrous or granular microstructure such as an intracellular chromosome, mitochondria, microtubule, actin bundle or the like, or a state where a microorgan is alive and moving.

  In addition to living organisms, it is also suitable for observing transparent crystals or amorphous substances such as glass and plastic.

  As an embodiment, there are two types of polarizing microscopes, an upright microscope and an inverted microscope, depending on whether the position of the light source is lower or upper. Examples of the light source include a laser light source, a mercury lamp, a xenon lamp, a metal halide lamp, and a halogen lamp. A crystal such as KDP can be used as a variable phase shifter as a liquid crystal plate or a Pockels cell.

  The polarizing microscope observation method and the polarizing microscope system according to the present invention can be used in various industrial fields.

  First, as described above, a biological sample can be dynamically observed including cells in a biological tissue in an unstained and alive state, or the internal structure of the cell. It is possible to identify the moving state of fibrous or granular microstructures such as chromosomes, mitochondria, microtubules, actin bundles, etc. in the cell, and microorgans with high spatial discrimination and high sensitivity. Image data can be collected. In addition, three-dimensional solid construction of cells and the like is possible by reducing the depth of focus and collecting information in the depth direction.

  Second, in the medical field such as livestock industry and obstetrics and gynecology, observation of the state where staining is not possible by cell manipulation / gene manipulation necessary for biotechnology such as fertilized egg / fission, nuclear transfer, etc.・ Because it is possible to see clear images in micro work, it is considered to make a great contribution.

  Thirdly, in the pharmaceutical industry, it is useful for fine static or dynamic observation of a drug in a crystal or thin film state of a colorless drug compound.

  Fourthly, it is useful for observing dynamic changes such as crystals caused by physical, chemical or biological treatments on transparent crystals or thin film crystals. Changes in crystals or the like can be observed when current, voltage, pressure, stress is applied, or dynamic changes due to melting or melting.

  Fifth, it is useful for observing static or dynamic changes with respect to the environmental conditions similar to those given in the fourth aspect, for an amorphous substance such as plastic and glass.

  As described above, the polarizing microscope observation method and the polarizing microscope system of the present invention have been described based on the principle and the examples, but the present invention is not limited to these and various modifications are possible.

It is a figure which shows the structure of one Example of the polarizing microscope system of this invention. It is a figure which shows typically the process of obtaining the retardation distribution image which changes temporally from the image obtained with a CCD camera by changing the phase difference given by a Pockels cell continuously and periodically.

Explanation of symbols

S ... Sample 1 ... Stage 2 ... Objective lens 3 ... CCD camera 4 ... Light source 5 ... Condenser 6 ... Linear polarizer (polarizer, polarizer)
7 ... Linear polarizer (analyzer, analyzer)
8 ... Interference filter 9 ... Pockels cell 10 ... Computer 11 ... Parallel port 12 ... Pockels cell controller 13 ... Monitor

Claims (9)

In an observation method using a polarization microscope in which a polarizer is arranged in a single-wavelength sample illumination optical path and an analyzer is arranged in a crossed Nicols state in the optical path from the objective lens to the imaging surface,
A variable phase shifter is arranged between the polarizer and the sample or between the sample and the analyzer, and the phase difference given by the variable phase shifter is set to three phase differences different from each other with time. A polarizing microscope observation method characterized in that one polarizing microscope image is taken and a phase difference distribution image of a sample is calculated from the obtained three images. The phase differences given by the variable phase shifters are θ ₁ , θ ₂ , and θ ₃ , respectively, and the light intensities on the imaging surface when the phase differences θ ₁ , θ ₂ , and θ _{3 are} given are I (x) ₁ , I ( The phase difference distribution image R (x) is calculated by calculating Equation (29) when x) ₂ and I (x) ₃ (x is a coordinate on the imaging surface). The polarizing microscope observation method according to 1.
R (x) = 1/2 ×
| {(Θ ₂ ² −θ ₁ ² ) −p (θ ₃ ² −θ ₁ ² )}
÷ {(θ ₂ −θ ₁ ) −p (θ ₃ −θ ₁ )} | (29)
However,
p = (I (x) ₂ −I (x) ₁ ) / (I (x) ₃ −I (x) ₁ ) (24)
It is. The polarizing microscope observation method according to claim 1, wherein the variable phase shifter includes a Pockels cell. The polarizing microscope observation method according to claim 1, wherein the variable phase shifter includes a liquid crystal element. The polarizing microscope observation method according to any one of claims 1 to 4, wherein a phase difference given by the variable phase shifter is 1/36 or less of a wavelength. _3. The polarization microscope observation method according to claim 2, wherein the phase difference given by the variable phase shifter is θ ₁ = 0, θ ₂ = a, θ ₃ = −a. The three different phase differences are periodically given, and the phase difference distribution image is taken by calculating each of the successive three images obtained by shifting the successive images taken at the respective phase differences by the photographing time interval. The polarizing microscope observation method according to any one of claims 1 to 6, wherein the calculation is continuously performed at each time interval. In a polarizing microscope system using a polarizing microscope in which a polarizer is arranged in a single-wavelength sample illumination optical path and an analyzer is arranged in a crossed Nicols state in the optical path from the objective lens to the imaging surface,
A variable phase shifter is disposed between the polarizer and the sample or between the sample and the analyzer;
Control means for causing the variable phase shifter to generate three phase differences different from each other over time; imaging means for capturing one polarization microscope image in synchronization with the variable phase shifter when each phase difference is applied; A polarization microscope system comprising: a calculation unit that calculates a phase difference distribution image of a sample from three captured polarization microscope images; and a display unit that displays the calculated phase difference distribution image of the sample. The control means controls the three different phase differences depending on the variable phase shifter to periodically change, and the imaging means synchronizes each phase difference of the variable phase shifter with one polarization microscope image. By taking one image of the phase difference distribution image of the sample from three consecutive polarized microscope images while shifting the time interval of changing the phase difference by the control means by the control means by the calculation means, 9. The polarizing microscope system according to claim 8, wherein phase difference distribution images are successively obtained at the same time interval.

Images