JP2007108664A5 - - Google Patents

Description

Asbestos-containing sample preparation method and asbestos observation method

  The present invention relates to a microscope for inspecting asbestos contained in a sample. Samples observed with the microscope of the present invention are those collected from dust and building materials in the atmosphere, and are mucous membranes and tissues collected from humans suspected of malignant mesothelioma caused by asbestos.

  As a method for inspecting the asbestos content of a sample, there is a disperse staining method using an optical microscope. The disperse dyeing method is an analysis method utilizing the fact that the asbestos exhibits a dispersion color peculiar to the type depending on the difference between the optical refractive index of asbestos and the refractive index of the immersion liquid in which asbestos is suspended.

  The optical system in the dispersion dyeing method will be described with reference to FIGS. In the condenser optical system in which light is incident on the specimen 101, a ring-shaped light beam is formed. For this purpose, light from the light source is converted into a parallel light beam 102 by a collimator lens, and a ring slit 103 centering on the optical axis of the parallel light beam is placed on the parallel light beam. The parallel light flux having the ring cross section thus obtained is collected by the lens 104 and is incident on the sample 101. Due to the difference in refractive index between the asbestos 111 and the immersion liquid 114, the light is divided into straight light and dispersion light that are not affected by changes in the propagation direction. The straight traveling light is blocked by the light blocking portion 106 disposed on the rear side of the lens 105. The ring slit 103 and the light-shielding part 106 are arranged at optically translated positions. On the other hand, the dispersed light is not blocked by the light shielding unit 106 but is imaged by the naked eye or an imaging device as observation light.

  The dispersion color due to the straight traveling light of the wavelength λ1 and the dispersed light of the wavelength λ2 depends on the refractive index of the immersion liquid 114. FIG. 12 shows the correspondence between the asbestos type, the refractive index of the immersion liquid, and the dispersion color.

  A method for preparing a specimen from a sample collected from building materials will be described. Place 10-20 mg of analytical sample and 40 ml of purified water into a 50 ml stoppered test tube, stir, then drop 10-20 μl onto a slide glass while stirring with a magnetic stirrer, and air dry, that is, leave to dry . Next, 3 to 4 drops of three types of immersion liquids with refractive indexes = 1.550, 1.680, and 1.700 are dropped onto each slide glass, mixed and dispersed with the immersion liquid at the tip of the tweezers, and then wiped on the cover glass The sample number and the refractive index of the immersion liquid are described.

  A method for preparing a specimen from a sample collected from the atmosphere will be described. The air is pumped and the filter paper is passed through to capture asbestos. The filter paper is clarified with dimethyl phthalate and diethyl oxalate, or acetone and triacetin.

  When using dimethyl phthalate and diethyl oxalate, add unused collection filter paper at a rate of 0.05 g / ml in a solution in which dimethyl phthalate and diethyl oxalate are mixed at a ratio of 1: 1. The filter paper is dropped almost on the center of the slide glass, and a filter paper that captures asbestos is placed thereon with the collection surface facing upward. When the filter paper becomes transparent, place the cover glass and fix it.

  When acetone and triacetin are used as reagents, a filter paper that captures asbestos is placed on a slide glass with the collection surface facing up, and acetone vapor generated by an acetone vapor generator is applied. When the filter paper becomes transparent, triacetin is dropped on the filter paper, and a cover glass is placed on the filter paper and fixed.

Specimens obtained by sampling from the atmosphere are observed with a phase contrast microscope. Although the optical elements to be used differ between the microscopic observation and the phase difference observation by the disperse staining method, the operations are similar and can be used in common. Observation methods can be easily changed by switching optical elements.
A sample collected from a living body may be observed. Tests are being conducted to see if asbestos was inhaled and entered the lungs, and the presence in the body. The autopsy lung and malignant mesothelioma tissue etc. was homogenized with pure water with a homogenizer, and specimens that air-dried and filtered through a filter. This sample is observed for phase difference.
JP 2000-88838 A

  In the disperse dyeing method, a disperse color is given by the difference in refractive index between the immersion liquid and asbestos, and it is determined whether the color is asbestos. However, depending on the crystal, it may not be possible to determine whether the dispersed color is thin and asbestos. If rock wool is mixed in the specimen in such a state, the discrimination becomes more difficult.

In the sample of the sample collected from the atmosphere, the transparent filter paper and asbestos are observed in the same visual field. At this time, the influence of the phase contrast caused by the filter paper fibers cannot be wiped off. If the contrast due to asbestos is small, that go out is necessary to eliminate the effect of filter paper fiber.

Since the polarization characteristic asbestos itself, be disposed a polarizing plate in the microscope beam path, the crystal being observed by the rotation of the polarizer is changed in color to observe if asbestos. On the other hand, since rock wool and filter paper fibers do not have polarization characteristics, the color does not change even when the polarizing plate is rotated.

  When the polarizing plate is rotated while observing with a microscope, the polarizing plate can be rotated and the asbestos with polarization characteristics can be observed by changing its color to distinguish it from other rock wool and filter paper fibers. can do. Therefore, it is necessary to avoid agglomeration of asbestos in specimen preparation.

  In view of these circumstances, the present invention has an object to provide a specimen preparation method and an observation method for efficiently observing asbestos contained in a sample with high detection sensitivity and ensuring the preservation of the sample and its data. To do.

  The specimen preparation method of the present invention applies ultrasonic vibration to an asbestos-containing sample suspension dropped on a slide glass.
  Thereby, the asbestos fiber which tends to aggregate can be separated into individual fiber particles.
  Moreover, the sample preparation method of this invention adds ethanol to the asbestos containing sample suspension liquid dripped on the slide glass.
  Thereby, the exposed state of the outermost surface of asbestos can be maintained.
  Next, in the observation method of the present invention, an asbestos-only region is designated on an image, and optical spectrum data of the designated region is obtained.
  Thereby, spectral data of only asbestos can be obtained.
  In the observation method of the present invention, marking indicating asbestos is performed on an image.
  Furthermore, in addition to the sample number, the observation method of the present invention includes asbestos sample collection information, the use address / use location / purpose of use, conditions from sample collection to slide glass sample observation, optical conditions, images, regions and The spectrum, aspect measurement conditions, total particle number / asbestos particle number, measurer, and sampler are described together with the observation result.
  This ensures data traceability.
  The observation method of the present invention describes the X-ray diffraction test result together with the observation result.
  Furthermore, the observation method of the present invention stores the X-ray diffraction test results together with the observation results.

  By utilizing this invention, the type and amount of asbestos contained in the sample can be efficiently observed with high detection sensitivity. In addition, data and samples and specimens can be stored without fail from sample collection. Furthermore, it is possible to re-inspect efficiently.

   FIG. 1 shows an example of the microscope 1 of the present invention. The microscope in this example is an upright microscope, but may be an inverted microscope having the same optical elements and functions. The light emitted from the light source 3 is collimated by the lens 4 to be collimated. This is reflected by the mirror 5 and bent upward, condensed by the window lens 6, and converted into parallel light by the lens 7 again. This parallel light has a beam diameter different from that of the parallel light produced by the lens 4. That is, it has a function of changing the beam diameter.

  The parallel light transmitted through the lens 7 is polarized and modulated by the polarizing plate 8. The polarizing plate 8 is rotatable and can change the polarization direction to an arbitrary direction. Further, the polarizing plate 8 is not necessarily between the lens 7 and the specimen 10. It may be on the light source side or sample side of the window lens 6 or between the objective lens after passing through the specimen and the prism 13. The number of polarizing plates is not necessarily one, and two polarizing plates may be provided so that one or both of them can be rotated so that the angle of the polarization direction can be changed mutually. The polarizing plate 8 may be rotatably disposed between the lens 7 as a condenser and the window lens 6. The configuration is simple, and the operability is good because the hand is not greatly separated from the desk, so the inspection efficiency is improved.

  The ring slit 16 used together with the dispersion objective lens is arranged at a position where it is collimated by the lens 7 to become parallel light. It may be in front of or behind the polarizing plate 8. The ring slit 16 may be selectively used depending on the magnification of the dispersion objective lens. However, it is not necessary to have the ring slits 16 corresponding to the objective lenses of all magnifications, and a single ring slit may be used for several objective lenses. The ring slit 16 may be used in common with the ring slit for a phase difference objective lens when performing phase difference observation. Light that has passed through the specimen 10 placed on the stage 9 passes through the objective lens 11. The stage 9 has a mechanism for rotating the specimen.

  Some objective lenses have different magnification and contrast forming functions. A bright field observation objective lens, a dark field observation objective lens, a phase difference objective lens, a dispersion dyeing objective lens, and the like. One or more of these objective lenses may be attached to the revolver 18 so that they can be exchanged and switched.

  The dispersion objective lens 11 needs to be sufficiently shielded by the light shielding portion of the objective lens. If one side of the plane parallel plate that forms the ring slit is light-shielding coated, defects in the coating, for example, pinholes, remain and become the background of the image. If a light-shielding coat, for example, a chrome coat, is applied to both sides of the plane-parallel plate, it is possible to sufficiently shield the light by preventing coating defects. Further, if both sides are shielded from light, the transmittance decreases accordingly, and it is effective for obtaining contrast. If it is difficult to sufficiently shield the light with a coat, a dispersion dyeing objective lens may be created by attaching a metal ring to a plane parallel plate.

  The light shielding part in the objective lens 11 is arranged at a position conjugate with the ring slit on the condenser side. On the other hand, the light shielding portion is not necessarily in the objective lens, and the pupil of the objective lens may be projected by a relay system and disposed at the projected position.

  The light transmitted through the objective lens 11 is observed through the eyepiece 14. Moreover, it images with the imaging device 15 via an imaging lens. The imaging device is, for example, a digital camera for a microscope. The captured image is recorded and stored in the computer or image processing apparatus via the imaging apparatus 15 itself, a storage medium, or communication means.

  In the microscope configured as described above, if the ring slit is shared, the disperse staining observation and the phase difference observation can be performed by switching the objective lens attached to the revolver. The same location on the specimen can be observed with both microscopic methods. In addition, polarization observation can be combined by arranging polarizing plates on the light source side and the opposite side of the specimen and rotating one or both of them. A polarizing plate arranged on the light source side of the specimen 10 is called a polarizer. A polarizing plate disposed between the specimen 10 and the lens barrel portion 17 is called an analyzer. In the polarization microscope observation, the analyzer may be rotated to be in a crossed Nicols state. Alternatively, the polarizer may be rotated to obtain a change in polarization direction relative to the polarization direction of the sample itself.

  The prism 13 has a polarization characteristic. For this reason, if the polarizing plate is placed in a microscope as it is, the brightness and color of the left and right images may not be the same. In particular, when asbestos is observed by rotating the polarizing plate, the depolarizing element 20 is disposed after the analyzer. Specifically, a quartz plate having a thickness of 1 mm or more is used. If a wave plate 19 such as a sensitive color plate or a quarter-λ plate is disposed between the depolarizing element 20 and the window lens 6, the contrast of the observation color in the polarization observation can be increased.

  Disperse staining microscope equipped with a polarizer and analyzer, rotate the polarizer or analyzer to distinguish between asbestos and rock wool, then remove the polarizing plate from the optical path and perform disperse staining observation and phase difference observation. Extraction and dispersion color can be measured. Similarly, only asbestos can be extracted and observed in phase difference observation. Even if the polarizing plate is not removed from the optical path, the disperse staining observation or the phase difference observation may be performed after adjusting the mutual angle so as to cancel the polarization of the light entering the eyepiece 14 or the imaging device 15.

  Generally, a white light source is used as the light source 3, but a light source that emits light in a spectral band necessary for obtaining a dispersed color or a light source that does not emit light in an unnecessary spectral band can be used. Light that is unnecessary for the disperse color causes an unnecessary background of the disperse color, so it is effective to remove it. Select from white light source by filter. When an electroluminescent element (hereinafter referred to as LED) is used as a light source, a luminescent color may be selected. In the LED, an element that does not emit green light that contributes little to the formation of dispersed colors, for example, emits light in the red and blue bands may be used.

  The specimen observed with the microscope of the present invention does not necessarily have to be a specimen prepared by the method described in the present specification.

  Next, an embodiment of the present invention relating to sample preparation used in this observation system will be described. In the process of preparing a specimen from a sample collected from a building material or the like, ultrasonic vibration is used when mixing 10 to 20 mg of the sample and 40 ml of purified water and stirring the liquid in which the sample is suspended. As shown in FIG. 3, the flask container 33 containing the suspension 34 is immersed in water 32 in the ultrasonic bath 31 and the ultrasonic vibration is propagated to the suspension and stirred. Thin fibers such as asbestos may be agglomerated with each other. In such a case, ultrasonic vibration can be applied to separate the individual fiber particles. After this, stirring is continued with a magnetic stirrer in order to break the aggregation again. Agitation by mechanical shaking may be included in these steps.

  Ultrasonic vibration is used after dripping the stirred suspension on the slide glass. The vibrator 42 is applied to the surface of the slide glass 44 opposite to the suspension 44 dropped on the slide glass 43, and the ultrasonic vibration from the vibration source 41 is propagated to the suspension. Depending on the shape of the tip of the vibrator, it can be a plane wave ultrasonic wave or can be concentrated in a specific region as a spherical wave. If a vibrator having a thin tip is used, ultrasonic vibrations may be generated by immersing the tip of the vibrator directly into a suspended liquid. When the concentration of asbestos is low, it is precipitated by centrifugation, and the concentration is increased by removing the supernatant. This makes it easier to search for asbestos within the microscope field of view.

  Once air-dried and the immersion liquid is dropped, if ethanol is dropped before air-drying and then air-dried, there is an effect of maintaining exposure of the outermost surface of asbestos. It also has the effect of speeding up drying. Then, immersion liquid is dripped. After the immersion liquid is dropped, the mixture is stirred again with the tip of a tweezers or the like. At this time, ultrasonic vibration is applied to the immersion liquid from the slide glass side or directly. Then, a cover glass is placed. The periphery of the cover glass is sealed with a sealant 25 as shown in FIG. By sealing, it can be re-observed as it is even after a long period of time or a long period of time. Even after sealing, stirring with ultrasonic waves is possible.

  The specimen prepared in this way is attached with a label 24 describing any of the sample number (hereinafter referred to as ID), the refractive index of the immersion liquid, the sample collection date, the material creation date, the approximate collection location, and the like. Some of these are useful for estimating the origin of the sample and help identify the sample during retesting. On the other hand, in order to avoid preconceptions, only the ID and the refractive index of the immersion liquid may be described.

  Next, an embodiment of the invention relating to acquisition and measurement of images and data in this observation system will be described. FIG. 5 shows a method for obtaining the ratio of the vertical and horizontal lengths of the asbestos 51 (hereinafter referred to as aspect ratio). The measurement process shown in FIG. 5 is performed using the acquired image or an image that has been stored once and reproduced.

   In FIG. 5A, a1 and a2 are designated with a cursor, and the ratio between the screen length obtained based on the total magnification of the image and the actual sample length is obtained. Using this, the distance α on the sample between a1 and a2 is measured. Similarly, β is measured by designating b1 and b2. α / β is the aspect ratio. There is also a method of determining the a1 and a2 and b1 and b2 while viewing the image by the measurer. A threshold value may be set based on the brightness and contrast at each coordinate in the image space, and the coordinates intersecting the threshold value may be used.

  Since β is often 1 μm or less, the measurement error tends to increase. Therefore, the method shown in FIG. 5B is effective. Asbestos has a straight fiber shape. On the image, c1 and c2 are designated, and a line perpendicular to the straight line c1c2 (hereinafter referred to as a perpendicular line) o1o2 is obtained. The intersection between the perpendicular line o1o2 and the straight line c1c2, that is, the distance between d1 and d2 shown in FIG. 5B is obtained.

  Based on the ratio between the length on the screen obtained based on the overall magnification of the image and the length on the actual sample, a reference length value and a scale bar corresponding to the length are displayed on the image. It should be noted that a1, a2, b1, b2, c1, c2, d1, d2, o1, o2 and the scale bar are stored as image-related data, reproduced and used.

  Asbestos with a length of 5 μm or more is considered harmful to the human body. Further, asbestos having an aspect ratio of 3 or more is regarded as a problem. Each asbestos obtained from one or more images is classified and stored and recorded. At that time, marking is performed to indicate which type of asbestos image belongs to. An example is shown in FIG. The asbestos 61 is marked with an arrow 63 and the non-asbestos material 65 is not marked. Of course, the material 65 may be provided with another marking indicating rock wool or other materials. Another type of asbestos is marked with another shaped arrow 64 as shown in FIG.

  A comment or the like is written in a space attached to the marking, and information on the asbestos can be obtained on the image. The comment space may be displayed outside the image on the display screen or on another screen without being shown on the image.

  In a specimen prepared from a sample collected from a living body, an asbestos body (referred to as asbestos body) shown in FIG. 7 is observed in addition to the asbestos fiber (referred to as naked fiber) shown in FIG. Asbestos is chemically and thermally stable. Moreover, it is excellent in electrical insulation. For this reason, asbestos remains in the body for a long time. Asbestos bodies are covered with asbestos and a substance mainly composed of mucopolysaccharide containing iron derived from blood. The length ranges from several μm to several hundred μm. The differences shown in FIGS. 7A, 7B, and 7C are produced depending on how they are covered. FIG. 7 (A) shows a dumpling shape at intervals. FIG. 7B shows a continuous dumpling shape. FIG. 7C shows a dumbbell shape.

  These images are measured and marked as shown in FIG. 5 and FIG. 6, and can be stored, recorded, and reproduced together with classification data based on shapes and measured values. By measuring the shape and average diameter of the asbestos body, it is possible to provide data for estimating the period after the asbestos enters the body.

  Stanton et al. Say that mesothelioma occurs not only asbestos, but also a thin, long fibrous substance that is stable in vivo, regardless of its chemical composition or crystal structure. For example, if silica fibers are recognized, it is useful as diagnostic information. Silica fibers can be observed with a polarizing microscope, a disperse dye microscope, or a combination thereof.

  Further, in a specimen prepared from a sample collected from a living body, an iron-containing body (referred to as a ferruginous body) may be observed together with the asbestos body shown in FIG. The ferruginous body is iron-stained and can be observed with a bright-field microscope.

  FIG. 8 shows an example of an image in which substances other than asbestos, for example, rock wool, overlap when measuring the dispersed color. In order to measure the spectrum of only asbestos 81, a region 83 where the substance 82 is not overlapped is specified on the image, and if the spectrum of this region is measured, asbestos-only data can be obtained. Slight changes can be measured. If it is determined as one asbestos crystal, a plurality of regions may be designated within that range.

  As shown in FIG. 9, when spectra acquired from images determined to be the same type of asbestos are arranged in the same graph and compared, the peak wavelength, peak value, and spread in the wavelength region can be compared.

  Next, we will explain how to store and report observation results, specimens, specimens, etc., and the counting system. Information and data related to the collected sample and a part of the collected sample to be stored are collectively managed. Information and data are stored and managed by a computer, and part of the collected sample and specimen are stored and taken in and out by a storage management system linked to this computer. All identification is done by sample number. The sample number need not be one, and two numbers may be used.

  Information about samples collected from the atmosphere and building materials needs to be made public. At this time, there are a case where information that can identify the disclosure destination is necessary, and a case where the disclosure destination is not identified based on the principle of disclosure to many people. Information traceability must be ensured for which organizations have made it public. In that case, the sample number is used. When the sample number is not known, the sample number can be narrowed down and specified by searching from the attribute information such as the collection location, the collection date and time, and the asbestos type.

  Sample attribute information that is stored and managed together with the sample number includes collection address, sampler, sample location, purpose of use, space size, conditions from sample collection to sample preparation, optical conditions, images, and areas. There are spectrum, aspect measurement conditions, total particle number / asbestos particle number, information and results regarding X-ray diffraction test, measurement organization, measurer, presence / absence of re-examination and identification information thereof.

  Information on animal experiments made for research purposes is not only the paper, but also the information sent from the testing institution, with the sample number attached, asbestos aspirated, the substance aspirated as a control, and the type of other comparative substances You can know the origin. Information similar to information about samples collected from the atmosphere and building materials is also saved, and can be replayed and searched.

  Information about samples taken from patients and other humans must be strictly controlled. When disclosure is necessary, it must be done after a method or procedure that complies with the law. The information to be managed with respect to such a sample includes the identification of the sample provider, the history, the medical history, etc., in addition to the same type of information regarding samples collected from the atmosphere and building materials and information regarding animal experiments. In particular, the career needs to know what kind of job at which address and office at which time, and what kind of job he / she lived in.

  The management method of these information, samples, and specimens can be standardized within an institution, nationwide, or internationally. Although all organizations can share, it may be shared within the scope of organizations that can cooperate. Since the information stored and managed may not necessarily be the same depending on the organization, the items to be shared may be determined as appropriate, or as many information items as possible may be set to allow some blanks.

It is a figure which shows the microscope by embodiment of this invention. It is a figure which shows the sample by embodiment of this invention. It is a figure which shows the state which gives the ultrasonic vibration by embodiment of this invention to a sample. It is a figure which shows the state which gives the ultrasonic vibration by embodiment of this invention to a sample. It is a figure which shows the display for measuring the aspect-ratio of asbestos by embodiment of this invention. It is a figure which shows the example which marks the asbestos by embodiment of this invention. It is a figure which shows the example of the shape of an asbestos body. It is a figure which shows the example which designates the area | region which measures the spectrum of the dispersed light of asbestos. It is a figure which shows the example of the spectrum of the dispersed light of asbestos. It is a figure which shows the principle of disperse dyeing | staining observation. It is a figure which shows generation | occurrence | production of the dispersed light by asbestos. It is a figure which shows the relationship between the kind of asbestos, the refractive index of immersion liquid, and a dispersion color.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Microscope 2 Mirror base 3 Light source 4 Lens 5 Mirror 6 Window lens 7 Lens 8 Polarizing plate 9 Stage 10 Sample 11 Objective lens 12 Polarizing plate 13 Prism 14 Eyepiece 15 Imaging device 16 Ring slit 17 Lens barrel part 18 Revolver 19 Wave plate 20 Depolarization element 21 Slide glass 22 Cover glass 23 Asbestos 24 Label 25 Sealant 31 Ultrasonic tank 32 Water 33 Flask container 34 Suspension liquid 41 Vibration source 42 Vibrator 43 Slide glass 44 Suspension liquid 51 Asbestos 61 Asbestos 62 Asbestos 63 Arrow 64 arrow 65 substance 81 asbestos 82 substance 83 area 101 specimen 102 parallel light beam 103 ring slit 104 lens 105 lens 106 light shielding part 107 dispersed light 111 asbestos 112 slide glass 113 cover glass 114 immersion liquid

Claims (6)

  In the optical observation of asbestos, an asbestos observation method characterized by designating a region containing only asbestos on an image and obtaining optical spectrum data of the designated region.   In the optical observation of asbestos, a marking showing asbestos on an image is performed.   In the optical observation of asbestos, in addition to the sample number, asbestos sample collection information includes the address used, place of use, purpose of use, conditions from sample collection to slide glass sample preparation, optical conditions, image, region and spectrum, An asbestos observation method, wherein the aspect measurement conditions, the total number of particles / asbestos particles, the measurer, and the sampler are described together with the observation result.   In the optical observation of asbestos, an asbestos observation method characterized by describing an X-ray diffraction test result together with an observation result.   In the optical observation of asbestos, an asbestos observation method characterized by storing an X-ray diffraction test result together with an observation result. Asbestos-containing sample creating method characterized that you added to ethanol to asbestos-containing sample-suspension liquid was dropped on a slide glass.