JP2019105703A - Phase difference image inspection device and phase difference image inspection method - Google Patents

Abstract

To provide an effective support means for a "functional diagnostic method" by using a phase difference microscope as a microinterferometer for non-staining real time inspection of living cells such as cancer cells and making use of superiority in a detail observation of its function by comparing it with a conventional inspection device.SOLUTION: A higher order light and a zeroth order light of a captured scattered diffraction light are continuously made to interfere with each other by a filter, and the observed data of the change phenomena including an extracellular appearance change phenomena (A) consisting of an extracellular appearance shape (A1) and the cell size (A2) of the cells that change every moment, and the intracellular structure change phenomena (B) consisting of an intracellular shape (B1) and the intracellular organelle size (B2) are acquired each time, and the interference image imaged on the basis of any change or change phenomena is obtained.SELECTED DRAWING: Figure 6

Description

  The present invention clearly visualizes fine phase differences almost invisible with a normal microscope of fine phase objects such as micro defects in the nuclei of living cells and glass plates and bubbles, and the shapes of phase objects and foreign substances in substances. Extracts, with a large field of view, microscopic anomalies such as distortions, refractive index changes, and micro cracks, enables inspection, and automatically identifies the shape of cells that are phase objects with nuclei inside And an inspection method and apparatus for identifying the type of the object to be inspected using the phase difference of the transparent object.

  Conventionally, observation and measurement of transparent particulate phase objects such as living cells have been performed using a phase contrast microscope [Non-Patent Document 1]. However, in the conventional phase contrast microscope, a ring diaphragm is inserted into the irradiation light, and it is narrowed with a condenser lens to create a narrow field of view, and a ring phase plate is placed on the back focal plane of the objective lens placed behind the sample. A complex optical system was used.

  Moreover, the conventional phase contrast microscope does not necessarily use a light source with coherency (coherence) as the light source, and the phases of the wave train of the light from the light source are aligned spatially and temporally. Absent. Therefore, since the interference fringes of the light wave fluctuate spatially and temporally, there is a disadvantage that the interference image of the phase difference object such as a nucleus in a living cell is not always clear.

  In addition, it is a phase contrast microscope [patent document 1] which uses a laser as a light source, and even if it is a phase contrast microscope having a light source in which wave series of irradiation light are uniform both spatially and temporally, the image is clear In a phase contrast microscope using a ring-shaped illumination [Patent Document 1], the field of view is narrow and shallow as in a conventional phase contrast microscope, and it is difficult to simultaneously observe a large number of fine objects in a wide field of view. Met.

In addition, there has been an attempt to remove the phase ring of the phase difference condenser section in order to secure a large degree of freedom in the measurement field of view compared to the conventional phase contrast microscope [Patent Document 2]. Because a ring-shaped illumination is used to sharpen the image, no phase contrast microscope with a wide and deep measurement field in the true sense is found, and any phase contrast microscope developed so far, The field of view was narrow and shallow, and it was difficult to observe many fine objects simultaneously in a wide field of view.

Furthermore, conventional phase contrast microscopes do not have the function of automatically measuring the number and behavior of plural shaped particle groups of different shapes including phase difference simultaneously for each same shape.

  The applicant of the present patent application has filed a patent application for an invention that solves such a problem, and has obtained patent 5733940 [patent document 3].

  In addition, the present applicant has filed a patent application for an invention that solves such a problem. Patent Document 4 is the subject patent application.

  Patent Document 5 discloses a cell image analysis apparatus capable of analyzing a plurality of generations of cell genes.

  In Patent Document 6, a phase-contrast image is obtained using a phase-contrast microscope, an edge is extracted from the phase-contrast image using a second-order differential filter, and the floating is performed based on the shape of the floating cell having the edge. Determining the activation state of the cells is described.

  Patent Document 7 describes that an omnifocal image generation unit that generates an omnifocal image as an original image is used to extract a neurite that appears in cells from the original image and determine the state of the neurite. Be done.

Patent No. 3455194 JP 2003-195180 A Patent No. 5733940 gazette JP, 2017-129760, A JP, 2012-39927, A Unexamined-Japanese-Patent No. 2008-212017 Patent No. 6015113

"Applied engineering 1", 3-8-2 phase difference method, (1999.7 publication)

Patent Document 3 discloses a laser light source, a laser beam expanding parallel light constructing optical system for forming a coherent expanding parallel laser beam, a Fourier transform lens disposed in the coherent parallel laser beam, and a front side of the Fourier transform lens Object introducing means provided, a phase filter provided on the back focal plane of the Fourier transform lens and passing a light diffraction pattern of the Fourier transform image, a back focal plane of the Fourier transform lens set as the front focal plane, and the phase filter An inverse Fourier transform lens that condenses high-order diffracted light and zero-order light that has passed through, and an electronic camera installed to capture an optical image formed on the condensing surface of the inverse Fourier transform lens. A phase difference image creating optical system for forming a phase difference object interference image of the introduced object projected from the object introducing means, which is installed on the optical axis,
In the phase difference image creating optical system, the Fourier transform lens is disposed in the coherent magnified parallel laser beam, and an image of a front focal plane introduced object of the Fourier transform lens is placed on a back focal plane of the inverse Fourier transform lens. At a position of the back focal plane to be imaged, a wide field of view is taken of the phase contrast microscope of the introduced object at a position on the optical axis on the front focal plane of the Fourier transform lens and outward outward in a direction perpendicular to the optical axis. A phase-contrast image display device is described.

  The present inventor has developed a phase contrast microscope, which is a useful technique that enables observation of transparent samples without staining, and the image outline is blurred, which is a drawback of conventional phase contrast microscopes. And (2) to solve the halo effect that is not present in the sample and to capture a phase image with a wider field of view and a deeper depth of field as compared to the conventional phase contrast microscope It was possible.

  In addition to the two problems described above, the conventional phase contrast microscope has a technical problem to be solved that (3) the contrast can not be optimized in accordance with different transmittances. In the technology described in the patent publication, no solution for the third technical problem is presented.

  The inventor of the present application holds a patent application that holds the optical system described in Patent Document 3 and simultaneously solves the three technical problems described above. It was published.

  When the phase-contrast microscope is used as a non-staining real-time inspection micro-interferometer for living cells such as cancer cells, its superiority in detailed observation of its function has become clear as compared with conventional inspection devices. So, it turned out that it could be an effective support tool for "functional diagnosis".

  In view of such a point, the present invention uses the phase contrast microscope as a microscopic interferometer for non-staining real-time examination of living cells, particularly proliferative cells such as cancer cells, in comparison with a conventional examination apparatus, and its function It is an object of the present invention to provide effective support means for the implementation of “functional diagnostic methods” by making use of the superiority in detail observation.

  In order to solve the problem, the sample is irradiated with a coherent laser beam with uniform intensity and parallel to the sample, high-order light of scattered diffraction light including fine image information is taken in, and among the scattered light, high-order light and zero-order light are optical A laser phase-contrast image acquisition microscope is used, which is capable of interference with a filter to acquire observation data of a minute part of a sample and acquiring an imaged interference image.

More specifically, a laser light source, a laser beam expanding parallel light constructing optical system for forming a coherent expanding parallel laser beam, Fourier transform lens means disposed in the coherent parallel laser beam, a front side of the Fourier transform lens Object introducing means provided in the phase filter, provided on the back focal plane of the Fourier transform lens, a phase filter for converting zero-order light of the light diffraction pattern of the Fourier transform image into linearly polarized light and converting high-order diffracted light into circularly polarized light Means, inverse Fourier transform lens means disposed with the back focal plane of the Fourier transform lens means as the front focal plane and condensing high-order diffracted light and zero-order light passed through the phase filter, in front of the Fourier transform lens means Or a polarizing plate placed behind, and an electronic camera placed to take an optical image formed on the light collecting surface of the inverse Fourier transform lens means. The phase difference object of the introduced object projected from the object introducing means with the transmitted zero-order light linearly polarized light and the high-order diffracted light circularly polarized light installed on the optical axis and adjusted in intensity difference by the polarizing plate It has a phase difference image creation optical system that forms an interference image,
In the phase difference image forming optical system, the Fourier transform lens means is disposed in the coherent magnified parallel laser beam, and an image of a front focal plane introduced object of the Fourier transform lens means is placed behind the inverse Fourier transform lens means. At the position of the back focal plane to be imaged on the focal plane, the intensity difference for the introduced object on the optical axis on the front focal plane of the Fourier transform lens means and at the position outwardly deviated perpendicular to the optical axis is A phase difference image inspection apparatus characterized in that the phase difference object interference image formed by the adjusted transmitted zero-order light linear polarization and the high-order circular polarization is captured in a wide field of view may be used.

The phase difference image inspection apparatus described above comprises a rotatable λ / 2 plate (π filter) in front of a laser beam expanding parallel beam forming optical system forming a coherent expanding parallel laser beam bundle. The observation method of living cells with an image acquisition microscope irradiates a sample with coherent laser light of uniform intensity and parallel to it, and captures even higher-order light of scattered diffraction light including fine image information, and among the scattered light, higher-order light and In an observation method of a living cell by a laser phase difference image acquisition microscope capable of acquiring observation data of a minute part of a sample by causing interference with zero-order light with an optical filter, and acquiring an imaged interference image,
The sample is a living cell and
The sample is irradiated with coherent laser light of uniform intensity and parallel to the cells to form an image, the cells displayed in the image are identified, and higher-order light of scattered diffraction light of the identified cells is also incorporated,
Among the scattered and diffracted light taken in, the filter sequentially interferes with the higher-order light and the zero-order light and changes the cell appearance of the cell (A) and the intracellular structure change (B) which change from moment to moment It is characterized in that observation data consisting of change phenomena that become

The observation method of a living cell using the laser phase contrast image acquisition microscope of the present invention irradiates a sample with coherent laser light of uniform intensity and parallel to it, takes in even higher-order light of scattered diffraction light including fine image information, and scatters it Among diffracted light, a laser phase difference image acquisition microscope capable of acquiring observation data of a minute part of a sample by causing high-order light and zero-order light to interfere with an optical filter, and acquiring an imaged interference image. In the observation method of the living cell used,
The sample is a living cell and
The sample is irradiated with coherent laser light of uniform intensity and parallel to the cells to form an image, the cells displayed in the image are identified, and higher-order light of scattered diffraction light of the identified cells is also incorporated,
Of the incorporated scattered diffraction light, a filter is used to continuously interfere with the high order light and the zero order light with a filter, and the cell appearance including the cell appearance shape (A1) and the cell size (A2) of the cell which changes from moment to moment Observation data consisting of the change phenomenon (B) including the change phenomenon (A) and the intracellular structure change phenomenon (B) including the intracellular shape (B1) and the intracellular organelle size (B2) are acquired from time to time, It is characterized by acquiring an intermittent interference image imaged based on any change phenomenon or all change phenomena to be combined.

  The observation method of a living cell using the laser phase contrast image acquisition microscope of the present invention is the above-mentioned observation method of a living cell using the laser phase contrast image acquisition microscope. Intracellular structure including the cell appearance change phenomenon (A) including the cell appearance shape (A1) and the cell size (A2) of the cells by comparison of images, and the intracellular structure including the intracellular shape (B1) and the intracellular organelle size (B2) It is characterized in that the change phenomenon (B) and the change phenomenon to become are obtained, and the cell type is identified from the change phenomenon.

The laser phase-contrast image acquisition microscope of the present invention irradiates a sample with coherent laser light of uniform intensity and in parallel, and captures even higher-order light of scattered diffraction light including fine image information, and among the scattered diffraction light, higher-order light and In a laser phase difference image acquisition microscope capable of acquiring observation data of a minute portion of a sample by causing interference with zero-order light with an optical filter, and acquiring an imaged interference image,
The sample is a living cell and
The sample is irradiated with coherent laser light of uniform intensity and parallel to the cells to form an image, the cells displayed in the image are identified, and higher-order light of scattered diffraction light of the identified cells is also incorporated,
Among the scattered and diffracted light taken in, the filter sequentially interferes with the higher-order light and the zero-order light and changes the cell appearance of the cell (A) and the intracellular structure change (B) which change from moment to moment It is characterized in that observation data consisting of change phenomena that become

The biological cell observation apparatus using the laser phase contrast image acquisition microscope of the present invention irradiates a sample with coherent laser light with uniform intensity and parallel, and takes in even higher-order light of scattered diffraction light including fine image information, Among the diffracted lights, a laser phase difference image acquisition microscope is used that can acquire observation data of a minute part of the sample by causing high-order light and zero-order light to interfere with each other with an optical filter. In the living cell observation device,
The sample is a living cell and
The laser phase contrast image acquisition microscope
The sample is irradiated with coherent laser light of uniform intensity and parallel to the cells to form an image, the cells displayed in the image are identified, and higher-order light of scattered diffraction light of the identified cells is also incorporated,
Of the incorporated scattered diffraction light, a filter is used to continuously interfere with the high order light and the zero order light with a filter, and the cell appearance including the cell appearance shape (A1) and the cell size (A2) of the cell which changes from moment to moment The observation data consisting of the change phenomenon (B) including the change phenomenon (A) and the intracellular structure change phenomenon (B) including the intracellular shape (B1) and the intracellular organelle size (B2) are acquired from time to time,
Means of observing living cells
It is characterized by acquiring an intermittent interference image imaged on the basis of any change phenomenon or all change phenomena in the combination.

  The biological cell observation apparatus using the laser phase contrast image acquisition microscope of the present invention is the biological cell observation apparatus using the above-described laser phase contrast image acquisition microscope, and it is Changes in cell appearance (A) including the cell appearance shape (A1) and cell size (A2) of the cells by comparison, and intracellular structure change phenomena including the intracellular shape (B1) and intracellular organelle size (B2) (B) It is characterized by acquiring the change phenomenon which becomes and identifying the cell type from the change phenomenon.

By using a laser phase contrast imaging microscope,
(1) By taking diffracted light having a wide area into high order light, contour definition is realized by fine image reconstruction by high order light.
(2) By providing the zero-order light transmission light transmitting means in the filter means, the zero-order light transmitted through the sample is prevented from causing self-interference, and the halo effect due to the effect of the zero-order light interference is prevented Can do.
(3) By adjusting the angle of the polarizing plate to attenuate the zero-order light and weaken the background light, the scattered light intensity is relatively strengthened, and the interference between the zero-order light background light and the high-order scattered light is It can enhance the contrast of the image.

  In this way, (1) the outline of the image is blurred and (2) the halo effect of the back light like pattern does not appear on the sample is solved, and the wide field of view compared to the conventional phase contrast microscope, A phase image having a deep depth of field can be captured, and (3) the optimization of contrast can be realized in accordance with different transmittances.

  Under such circumstances, according to the present invention, the observation method of a living cell using a laser phase contrast image acquisition microscope is an appearance shape of the cell by comparing interference images before and after timely interference images A1) Cell appearance change phenomenon (A) including cell size (A2), intracellular structure change phenomenon (B) including intracellular shape (B1) and intracellular organelle size (B2), nuclear shape (A) It is possible to obtain a change phenomenon consisting of nuclear structural change phenomenon (C) including C1) and nuclear organelle size (C2).

  As described above, according to the present invention, the phase contrast microscope is used as a microscopic interferometer for non-staining real-time examination of living cells such as cancer cells, and compared with the conventional examination apparatus in detail observation of its function. It is possible to provide an effective means of supporting “functional diagnostic methods” by taking advantage of its superiority.

The figure which shows schematic structure of the phase difference image display apparatus used for the Example of this invention. The enlarged view of the top of the drawings shown in FIG. 1 The figure which shows schematic structure of the phase difference image display apparatus described in the reference 3 The figure which shows the comparison of the microscope image of the glass fragment acquired by the phase difference image inspection apparatus used for the Example of this invention, and the phase difference image inspection apparatus described in the cited reference 3 The figure which shows typically the interference image 200 imaged based on the change phenomenon Diagram showing the situation where observation data consisting of change phenomena are acquired momentarily A diagram showing an example in which changes before and after change are acquired and displayed as interference images from moment to moment Figure showing an overall view image of observed lung cancer cells The figure which took out and showed the condition where the whole view image of FIG. 8 changes momentarily. Figure showing an image displayed from observation data that captured cancer cell shape change and cell nucleus shape change The figure which took out and showed the situation where the observation data in Figure 10 changes from moment to moment Figure showing an image displayed from observation data that captures changes in cancer cell shape and changes in cell nuclear shape and nuclear organelles The figure which took out and showed the situation where the observation data in Figure 12 changes from moment to moment Figure showing an image displayed from observation data that captures changes in cancer cell shape and changes in cell nucleus shape and organelle changes The figure which took out and showed the situation where the observation data of FIG. 14 changes every moment

  Hereinafter, a living cell observation apparatus using a laser phase contrast image acquisition microscope according to an embodiment of the present invention will be described.

  Before describing a living cell observation apparatus using a laser phase contrast image acquisition microscope, the laser phase contrast image acquisition microscope will be described.

  The laser phase difference image acquisition microscope is a phase filter means for converting zero-order light of the light diffraction pattern of the Fourier transform image into linearly polarized light and converting high-order diffracted light into circularly polarized light and transmitting it, for example, zero-order at the center of the Fourier transform plane. By placing a λ / 4 plate (π / 2 filter) with holes for light transmission and placing a polarizing plate in front of the camera, zero-order transmitted light from the sample and λ / 4 plate transmission can be obtained with the λ / 4 plate For example, the polarizing plate is produced by causing a phase difference with high-order diffracted light and causing an intensity difference between zero-order transmitted light (zero-order linearly polarized light) and high-order diffracted transmitted light (high-order circularly polarized light) with a polarizing plate. Arbitrary rotation makes it possible to adjust the angle and arbitrarily change the contrast of the image, and to adjust by relatively increasing the intensity of the high-order diffracted transmitted light to intensify their interference as well as the high-order diffraction Increase the contribution of micro-imaging of transmitted light for a wide field of view Characterized in that it clearly image the contours of such elaborate sample outline and the internal nucleus Ri.

  FIG. 1 shows an overall outline of a phase difference image display apparatus 100 possessed by the laser phase difference image acquisition microscope. FIG. 2 is an enlarged view of the image plane position of FIG. A detailed description of the enlarged view is given in the figure.

  FIG. 3 shows a phase difference image display device 100A described in Patent Document 3 which is presented in contrast for easy understanding of the phase difference image display device 100 shown in FIG.

  In the phase difference image inspection apparatus 100 (also referred to as a phase difference image display apparatus) 100 shown in FIG. 1 and the phase difference image inspection apparatus 100A shown in FIG. It is irradiated as a parallel luminous flux through 3. If the object to be inspected is placed in the object introducing means 6 disposed in front of the Fourier transform lens (Fourier transform lens means, the same applies hereinafter) 7 in the coherent collimated light beam or near the front focal plane of the Fourier transform lens 7 The Fourier transform image (light diffraction pattern) is formed on the back focal plane of the Fourier transform lens 7.

  In the phase difference image inspection apparatus 100A of FIG. 3, the zero-order light of the Fourier transform image is passed through the phase plate (λ / 4 plate) 20, through the light reduction filter for adjusting the light intensity, and after the Fourier transform lens 7. When passing through the inverse Fourier transform lens 9 installed with the focal plane as the front focal plane, the inverse Fourier transformed object light and the zero-order reference beam which is phase-transformed and reduced to parallel light are made of the inverse Fourier transform lens 9 An interference image of the phase difference object is captured by the electronic camera 10 on the light collecting surface.

  Since the technique shown in FIG. 3 uses coherent parallel laser light as the irradiation light of the inspection object, the inspection method becomes extremely simple, and as in the conventional method, after the ring diaphragm of the irradiation light and the condenser lens No effort is required to align the ring-shaped phase plate in the focal plane. Further, in the present technology, the zero-order light of the Fourier transform image is surely the reference beam of the phase image only by placing the phase plate and the attenuation filter on the back focal plane of the Fourier transform lens, and the phase difference of the phase object is The present technology has a simplification effect of the phase image because it can be converted to light intensity image information.

  Even in the phase difference image inspection apparatus 100 shown in FIG. 1, the phase plate (λ / 4 plate) 20, the inverse Fourier transform lens 9 and the electronic camera 10 are provided to achieve the above-described effect. The phase difference image inspection apparatus 100 includes a phase plate (λ / 4 plate) 20 and a polarizing plate 23 as a pair filter. With this configuration, the embodiment achieves the effect of sharpening the phase image. Furthermore, a rotatable λ / 2 plate (π filter) 21 is combined. Usually, circularly polarized light and linearly polarized light do not interfere, and light waves of the same polarization plane interfere only after the polarizing plate is provided. By this configuration, the embodiment achieves the effect of further clarifying the phase image. In addition, circularly polarized light having another polarization plane of high-order light contributes to clarification of detailed information of an object.

  In FIG. 1, a phase difference image inspection apparatus 100 includes a laser light source 1, a rotatable λ / 2 plate (π filter) 21, a laser light expansion parallel light configuration optical system that forms a coherent expansion parallel laser light flux, and a coherence Transform lens 7 installed in a collimated parallel laser beam, object introducing means 6 provided on the front side of this Fourier transform lens, zero on the light focal pattern of the Fourier transform image provided on the back focal plane of this Fourier transform lens A λ / 4 plate (π / 2 filter) phase filter 20 having a hole 22 at the center for passing only the next light and changing high-order diffracted light into circularly polarized light and transmitting it, and setting the back focal plane of the Fourier transform lens as the front focal plane Fourier transform lens 9 for collecting high-order diffracted light and zero-order light that has passed through the phase filter 20, and a rotatable polarization installed in front of or behind this Fourier transform lens 7 Each of the plate 23 and an electronic camera installed to capture an optical image formed on the light collecting surface of the inverse Fourier transform lens is projected on the optical axis and projected from the object introducing means 6 A phase difference image forming optical system for forming a phase difference object interference image of the introduced object.

In this phase difference image forming optical system, a Fourier transform lens 7 is disposed in the coherent magnifying parallel laser beam, and an image of a front focal plane introduced object of the Fourier transform lens 7 is a back focal plane of the inverse Fourier transform lens 9 The phase difference image of the introduction object at a position on the optical axis on the front focal plane of the Fourier transform lens 7 and outside at a right angle to the optical axis at the position of the back focal plane to be imaged on the electronic camera 10 You can shoot with.
The λ / 4 plate (π / 2 filter) phase filter 20 is typically provided at the back focal plane of the Fourier transform lens 7 and has a hole 22 at the center for passing zero-order light of the light diffraction pattern of the Fourier transform image. The second order diffracted light is changed to circularly polarized light and transmitted. Although it is possible to block the hole 22 with a light reduction filter to reduce the zero-order light, it is preferable to use a hole in order to change the intensity of the zero-order light by changing the intensity of the light reduction filter continuously. Thus, the holes in the present embodiment mean not only physical holes but also means that have no obstacle in passing zero-order light.

  A polarizing plate 23 installed in front of or behind the Fourier transform lens 7 is projected from the object introducing means by the transmitted zero-order light and the high-order diffracted light, which are installed on the optical axis and whose intensity difference is adjusted. A phase difference object interference image of the introduced object is formed. The intensity ratio between the zero-order light and the high-order light can be changed continuously.

  On the optical axis on the front focal plane of the Fourier transform lens 7 at the position of the back focal plane where the image of the front focal plane introduced object of the Fourier transform lens 7 is imaged on the back focal plane of the inverse Fourier transform lens 9 And the phase difference object interference image formed by the transmitted zero-order light and the high-order diffracted light of which the intensity difference is adjusted, in a wide view, for an introduced object at a position displaced outward in a direction perpendicular to the optical axis Incorporate.

  As described above, the rotatable λ / 2 plate (π filter) 21 is provided behind the laser light source 1 of the laser light expanding parallel light constructing optical system forming the coherent expanding parallel laser light flux. The angle is adjusted by rotating. The λ / 2 plate 21 placed behind the laser light source 1 functions to change the polarization plane of the linearly polarized laser light (change the angle of the polarization plane of the linear polarization). Although the feature of the present invention can be obtained without the λ / 2 plate, the arrangement of the λ / 2 plate 21 can provide an effective function when observing a sample having polarization dependency.

  As described above, the polarizing plate 23 is rotatable, and the angle is adjusted by rotating.

  The installation position of the perforated λ / 4 plate is the back focal plane of the Fourier transform lens 7, and the arrangement position of the polarizing plate 23 is either after the perforated λ / 4 plate before or after the inverse Fourier transform lens 9 Good.

  In the technique described in Patent Document 3, interference with scattered light is achieved by passing zero-order light through a λ / 4 plate and reducing light with a light reduction filter, but in the present embodiment λ By making a hole 22 for zero-order light transmission at the center of the 4th plate and placing a polarizing plate 23 in front of the camera 10, the scattered high-order light passes through the λ / 4 plate contrary to the technique described in Patent Document 3. It interferes with the zero-order light which is attenuated by passing through the polarizing plate 23.

  The λ / 2 plate (π filter) 21 forms a polarization plane by a π filter, and irradiation light or zero-order light (red display) and high-order diffracted light (blue display) are generated by rotation of the λ / 2 plate (π filter) 21. ) Is polarized in the polarization plane direction.

  Zero-order linear polarization and high-order diffracted circular polarization passing through the sample are formed.

  The high-order diffracted circularly polarized light (blue display) and the zero-order linearly polarized light (red display) transmitted through the π / 2 filter have the form shown in the figure.

  Now, it is assumed that the angle of the polarizing plate 23 is adjusted to the position (black display) shown in the figure.

  In the figure, the form state of the transmitted light transmitted through the filter pair consisting of the perforated λ / 4 plate 20 and the angle adjustable polarizing plate 23 is displayed in three colors of black, red and blue. By the action of the deflecting plate 23, the direction of the polarized light transmitting surface (black display in the figure) of the polarizing plate and the intensity of the same direction polarized transmitted light of high-order light of circularly polarized light (blue display) are shown by vectors, The contribution of the micro-imaging of the fold light is enhanced.

  Here, since the scattered high-order light passing through the λ / 4 plate is circularly polarized and has less attenuation at the polarizing plate, there is the advantage of simplicity that there is no need to finely process the polarizing plate 23 according to the aperture of the zero-order light. can get.

The following advantages can be obtained by using a filter pair consisting of the perforated λ / 4 plate 20 and the polarization adjustable polarization plate 23.
(1) By taking diffracted light into high-order light with a wide λ / 4 plate, contour clarification is realized by fine image reconstruction by high-order light.
(2) By opening a zero-order light transmission hole at the center of the λ / 4 plate, it is possible to prevent the zero-order light transmitted through the sample from causing self-interference, and the back light effect Halo effect can be prevented.
(3) By adjusting the angle of the polarizing plate to attenuate the zero-order light and weaken the background light, the scattered light intensity is relatively strengthened, and the interference between the zero-order light background light and the high-order scattered light is The image can be intensified, the image contrast can be intensified, and not only the outline of the object but also the internal structure can be made clear.

  FIG. 4 shows a comparison of microscopic images (FIG. 4 (b)) of glass fragments obtained by the phase difference imaging apparatus 100A described in the phase difference image inspection apparatus 100 (FIG. 4 (a)) and reference 3 FIG.

  In the image obtained according to the present embodiment, the thickness information of the inclined portion of the glass fragments appears as contour lines as shown in the figure, and the phase difference information is recorded by the phase difference image inspection apparatus described in reference 3 Not only the contour but also the interior clearly appears as compared to the microscopic image of the glass fragments obtained.

  In addition, it is shown that the minute information of the transparent object becomes clear in detail, and the acquired image is qualitatively different from the microscopic image of the glass fragments acquired by the phase-contrast image inspection device described in reference 3 ing.

  In Fig. 1 and Fig. 2, instead of 2 λ / 2 plate (π filter), λ / 4 plate (π / 2 filter) is used, and instead of perforated λ / 4 plate (π / 2 filter), glass plate is used The same effect as the above-described effect can also be produced by combining a phase filter having a circular λ / 4 plate with a size through which only zero-order light passes in the center. Also in this combined filter, the high-order diffracted light is circularly polarized and the zero-order light is linearly polarized, so the polarizing plate 23 installed in front of the camera produces the same effect. That is, a linear polarization filter means for zero-order light with zero-order light as linear polarization and a high-order diffracted light circular polarization filter means with high-order diffracted light as circular polarization are provided.

  This embodiment is also applied to polystyrene latex 20.3 μm, 10.3 μm and 5.0 μm standard particle groups, and the microscope image obtained by the phase-contrast image inspection device whose edge of the particle image is described in reference 3 It clearly appeared in comparison.

  As described above, simultaneous and real-time identification of a plurality of shapes is possible.

In a method of observing a living cell using a laser phase contrast imaging microscope,
The sample is a living cell and
The sample is irradiated with coherent laser light of uniform intensity and parallel to the cells to form an image, the cells displayed in the image are identified, and higher-order light of scattered diffraction light of the identified cells is also incorporated,
Of the incorporated scattered diffraction light, a filter is used to continuously interfere with the high order light and the zero order light with a filter, and the cell appearance including the cell appearance shape (A1) and the cell size (A2) of the cell which changes from moment to moment Observation data consisting of the change phenomenon (B) including the change phenomenon (A) and the intracellular structure change phenomenon (B) including the intracellular shape (B1) and the intracellular organelle size (B2) are acquired from time to time, A point-in-time interference image is acquired which is imaged on the basis of any or all of the combination phenomena being combined.

  By using the laser phase contrast image acquisition microscope, among the captured scattered diffraction light, the high-order light and the zero-order light are continuously interfered with a filter, and the cell external appearance shape of the cell changes from moment to moment ((2) A1) The cell appearance change phenomenon (A) including the cell size (A2), and the intracellular change phenomenon (B) including the intracellular shape (B1) and the intracellular organelle size (B2) It turned out that the observation data consisting of can be acquired every moment.

  FIG. 5 is a view schematically showing an interference image 200 imaged based on the change phenomenon.

  In FIG. 5, the change phenomenon includes the cell appearance change phenomenon (A) including the cell appearance shape (A1) and the cell size (A2) of the cells, the intracellular shape (B1) and the intracellular organelle size (B2). It consists of a change phenomenon that becomes an intracellular structural change phenomenon (B), and so on.

In FIG. 5, as a cell appearance shape (A1), new process generation
Cell size change as detail size (A2)
As intracellular shape (B1), new membrane development, new nuclear endometrial development, new organelle development,
Decreased intracellular organelles, nuclear organelle shape change
As organelle size (B2), organelle size change, nuclear organelle size
Change is shown. By using the said laser phase contrast image acquisition microscope, these change phenomena which combined the cell external appearance change phenomenon (A) and the intracellular structure change phenomenon (B) can be observed.

FIG. 6 shows the change in the cell appearance (A) and the intracellular structure change of the cells, in which the higher-order light and the zero-order light are continuously interfered with each other by the filter among the scattered and scattered light taken up. It is a figure which shows the condition which acquires observation data which consist of a change phenomenon used as a phenomenon (B) momentarily.
As shown in FIGS. 6 (i) to (v), the cell appearance and the intracellular structure of the cells change from time to time. Such change phenomena include cell appearance change phenomenon (A) including cell appearance shape (A1) and cell size (A2), and cells including intracellular shape (B1) and intracellular organelle size (B2) It is observed as an internal structure change phenomenon (B) and acquired as observation data. The acquired observation data is transmitted to a computer, for example, a personal computer, provided in addition to the laser phase difference image acquisition microscope, and displayed as an interference image immediately captured on the screen of the imaging device.

  When displaying as an interference image immediately captured on the screen of the image device, it is possible to display on the screen by deleting a part of the dry image using the image deleting means according to the needs of the observer. Observation data consisting of change phenomena can be acquired momentarily, and momentary interference images imaged on the basis of any change phenomenon or all alteration phenomena in the combination can be acquired.

  FIG. 7 shows a cell appearance change phenomenon (A) including cell appearance shape (A1) and cell size (A2), and an intracellular structure change phenomenon (cell shape (B1) and intracellular organelle size (B2). An example is shown in which the variation before and after the change is acquired and displayed as an interference image from moment to moment about the phenomenon that changes from moment to moment in B). In these examples, neonatal development and organelle size change were extracted and displayed in the upper figure. In the figure below, the occurrence of new projections as cell appearance shape change was extracted and displayed. If both of these changes occur, these changes are combined and displayed on the screen.

  Cell appearance change phenomenon (A) including the cell appearance shape (A1) and cell size (A2) of the cell by comparison of interference images before and after from time to time, intracellular shape (B1) and cells It becomes possible to obtain the change phenomenon that becomes the intracellular structural change phenomenon (B) including the organelle size (B2) and the change phenomenon and to identify the cell type from the change phenomenon.

  In the case of a sample cancer living cell, a cell appearance change phenomenon (A) including the cell appearance shape (A1) and the cell size (A2) of the cell by comparison of interference images before and after in time Obtain a change phenomenon that becomes an intracellular structure change phenomenon (B) including an intracellular shape (B1) and an intracellular organelle size (B2), and from the change phenomenon, cell proliferation specified by the change phenomenon is obtained. It can contribute to identifying the type of intense cancer cells.

Cell appearance change phenomenon (A) including the cell appearance shape (A1) and cell size (A2) of the cells, and intracellular structure change phenomenon (B) including the intracellular shape (B1) and the intracellular organelle size (B2) 2.) It is divided into two, but the intracellular structural change phenomenon (B) can be subdivided into an intracellular structural change phenomenon and a nuclear structural change phenomenon.

  FIGS. 8 to 15 are images capturing changes in observed lung cancer cells. The sample is a living cell of lung cancer. Although the sample is not specified as a lung cancer biological cell and is particularly effective for a sample that requires detailed observation of proliferative cells, the sample is specified and described as a lung cancer biological cell.

  FIG. 8 shows a whole field image of observed lung cancer cells.

  FIG. 9 is a diagram showing a situation in which the entire view image of FIG. 8 changes from moment to moment.

  FIG. 10 shows an image displayed from observation data capturing cancer cell shape change and cell nucleus shape change.

  FIG. 11 is a diagram showing the situation where the observation data of FIG. 10 changes momentarily.

  FIG. 12 shows an image displayed from observation data capturing changes in cancer cell shape and changes in the shape of the cell nucleus and organelles in the nucleus.

  FIG. 13 is a diagram showing the situation where the observation data of FIG. 12 changes every moment.

  FIG. 14 is an observation that captures changes in the shape of cancer cells and changes in the shape of the cell nucleus and changes in the shape of the cell nuclei and organelles in the other cells of the plurality of cells identified in the entire visual field image and present in the extracted image area Shows the image displayed from the data.

  FIG. 15 is a diagram showing a situation in which the observation data of FIG. 14 changes momentarily.

  Thus, when viewing FIG. 8, it is recommended to view the two figures in pairs in the manner referring to FIG. The same applies below. Also, when the image of FIG. 8 is acquired, the image shown in FIG. 9 is easily acquired by the image processing software.

  In FIGS. 8 and 9, coherent laser light having a uniform intensity and parallel to the cells is irradiated to a lung cancer living cell as a sample to form a whole field image, and some of the plurality of cells displayed in the image The area containing the cells of was identified as the observation area. The observation area is indicated by a frame on the drawing.

  In FIG. 10 and FIG. 11, the interference image which captured the cancer cell shape change and the cell-nucleus shape change was displayed on the screen. In the upper left, these changes are shown schematically. As can be seen from these screens, not only cancer cell shape change but also change in cell size are displayed as cell appearance change phenomenon (A) on one screen, and further as intracellular structure change phenomenon (B) Cell nucleus shape change interference image was displayed.

  In FIG. 12 and FIG. 13, the interference image which captured the change of the cancer cell shape, the shape of the cell nucleus, and the change of the nuclear organelle was displayed on the screen. In the upper left, these changes are shown schematically. As can be seen from these screens, on one screen, not only cancer cell shape change but also change showing the situation of increased cell size are displayed as cell appearance change phenomenon (A), and further intracellular structure change As the phenomenon (B), the shape of the cell nucleus and the nuclear organelle change interference image were displayed.

  In FIG. 14 and FIG. 15, the interference image which displayed the change of a cancer cell shape, the shape of a cell nucleus, and the change of the nuclear organelle about the other cell was displayed on the screen. In the upper left, these changes are shown schematically. As can be seen from these screens, not only cancer cell shape change but also cell size change is displayed on one screen as cell appearance change phenomenon (A) for the other cell, and further intracellular structure change As the phenomenon (B), the shape of the cell nucleus and the nuclear organelle change interference image were displayed. As shown in FIG. 11, not only cancer cell shape change but also change in cell size are displayed as cell appearance change phenomenon (A) for a plurality of cells on one screen, and further intracellular structural change As the phenomenon (B), it is possible to display a shape of a cell nucleus and a nuclear organelle change interference image. An interference image is provided which can be observed at one time to the inside of the cell in a wide area.

As can be seen from these images, according to the present embodiment, it is possible to image the active movement of cancer cells in the sample tissue in real time from moment to moment without requiring pretreatment. A support screen is provided for the rapid and clear diagnosis of cancer diseases. This support screen enables non-staining real-time examination every moment and enables functional diagnosis. The determination of cancer cells in situ can be made, and the time required for the determination may be several minutes to one hour, which contributes to a short time diagnosis.

  At the time of implementation of this embodiment, it is possible to obtain observation images of the cells from the momentary movement of real time of the cells, the cell nucleus and the subcellular organelle without requiring processing such as staining and thinning of the living cells, especially It is possible to acquire an observation image of a cancer cell with strong movement by proliferation. As described above, according to the present invention, the phase contrast microscope is used as a microscopic interferometer for non-staining real-time examination of living cells such as cancer cells, and compared with the conventional examination apparatus in detail observation of its function. It is possible to provide an effective means of supporting “functional diagnostic methods” by taking advantage of its superiority.

1: Light source (laser)
2: Lens 3: Lens 6: Object introduction means 7: Fourier transform lens 9: Inverse Fourier transform lens 10: Electronic camera or CCD camera 20: λ / 4 plate (π / 2 filter) phase filter (phase filter means)
21: λ / 2 plate (π filter)
22: hole 23: polarizing plate

Claims (6)

The sample is irradiated with coherent laser light of uniform intensity and parallel to the sample, high-order light of the scattered diffraction light including fine image information is also taken in, and the high-order light and the zero-order light among the scattered diffraction light are interfered by the optical filter. In an observation method of a living cell by a laser phase contrast image acquisition microscope capable of acquiring observation data of a minute part of a sample and acquiring an imaged interference image,
The sample is a living cell and
The sample is irradiated with coherent laser light of uniform intensity and parallel to the cells to form an image, the cells displayed in the image are identified, and higher-order light of scattered diffraction light of the identified cells is also incorporated,
Among the scattered and diffracted light taken in, the filter sequentially interferes with the higher-order light and the zero-order light and changes the cell appearance of the cell (A) and the intracellular structure change (B) which change from moment to moment The observation method of biological cells with a laser phase contrast image acquisition microscope characterized by acquiring observation data consisting of change phenomena every moment. The sample is irradiated with coherent laser light of uniform intensity and parallel to the sample, high-order light of the scattered diffraction light including fine image information is also taken in, and the high-order light and the zero-order light among the scattered diffraction light are interfered by the optical filter. In a method of observing a living cell using a laser phase contrast image acquisition microscope capable of acquiring observation data of a minute part of a sample and acquiring an imaged interference image,
The sample is a living cell and
The sample is irradiated with coherent laser light of uniform intensity and parallel to the cells to form an image, the cells displayed in the image are identified, and higher-order light of scattered diffraction light of the identified cells is also incorporated,
Of the incorporated scattered diffraction light, a filter is used to continuously interfere with the high order light and the zero order light with a filter, and the cell appearance including the cell appearance shape (A1) and the cell size (A2) of the cell which changes from moment to moment Observation data consisting of the change phenomenon (B) including the change phenomenon (A) and the intracellular structure change phenomenon (B) including the intracellular shape (B1) and the intracellular organelle size (B2) are acquired from time to time, A method of observing a living cell using a laser phase contrast image acquisition microscope, which comprises acquiring an intermittent interference image imaged on the basis of any change phenomenon or all change phenomena to be combined.   The observation method of a living cell using a laser phase contrast image acquisition microscope according to claim 2, wherein the living cancer cell is a sample cancer living cell, wherein said interference cell is compared with an interference image every moment by comparing the interference image before and after in time. Cell appearance change phenomenon (A) including cell appearance shape (A1) and cell size (A2), and intracellular structure change phenomenon (B) including intracellular shape (B1) and intracellular organelle size (B2) A method of observing a living cell using a laser phase contrast image acquisition microscope, which comprises obtaining a change phenomenon and identifying the type of cancer cell identified by the change phenomenon from the change phenomenon. The sample is irradiated with coherent laser light of uniform intensity and parallel to the sample, high-order light of the scattered diffraction light including fine image information is also taken in, and the high-order light and the zero-order light among the scattered diffraction light are interfered by the optical filter. In a laser phase difference image acquisition microscope capable of acquiring observation data of a minute part of a sample and acquiring an imaged interference image,
The sample is a living cell and
The sample is irradiated with coherent laser light of uniform intensity and parallel to the cells to form an image, the cells displayed in the image are identified, and higher-order light of scattered diffraction light of the identified cells is also incorporated,
Among the scattered and diffracted light taken in, the filter sequentially interferes with the higher-order light and the zero-order light and changes the cell appearance of the cell (A) and the intracellular structure change (B) which change from moment to moment A laser phase-contrast image acquisition microscope characterized by acquiring observation data consisting of change phenomena every moment. The sample is irradiated with coherent laser light of uniform intensity and parallel to the sample, high-order light of the scattered diffraction light including fine image information is also taken in, and the high-order light and the zero-order light among the scattered diffraction light are interfered by the optical filter. In a biological cell observation apparatus using a laser phase contrast image acquisition microscope capable of acquiring observation data of a minute part of a sample and acquiring an imaged interference image,
The sample is a living cell and
The laser phase contrast image acquisition microscope
The sample is irradiated with coherent laser light of uniform intensity and parallel to the cells to form an image, the cells displayed in the image are identified, and higher-order light of scattered diffraction light of the identified cells is also incorporated,
Of the incorporated scattered diffraction light, a filter is used to continuously interfere with the high order light and the zero order light with a filter, and the cell appearance including the cell appearance shape (A1) and the cell size (A2) of the cell which changes from moment to moment The observation data consisting of the change phenomenon (B) including the change phenomenon (A) and the intracellular structure change phenomenon (B) including the intracellular shape (B1) and the intracellular organelle size (B2) are acquired from time to time,
Means of observing living cells
An organism cell observation device using a laser phase contrast image acquisition microscope, characterized in that an interference image is acquired momentarily which is imaged based on any change phenomenon or all change phenomena which become the combination.   In the biological cell observation apparatus using the laser phase contrast image acquisition microscope according to claim 5, a cell appearance shape (A1) and a cell size of the cell by comparison of interference images before and after in time. Acquire the change phenomenon that becomes the cell appearance change phenomenon (A) including (A2), the intracellular structure change phenomenon (B) including the intracellular shape (B1) and the intracellular organelle size (B2), An organism cell observation device using a laser phase contrast image acquisition microscope characterized by identifying a cell type from a change phenomenon.

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