JP2006047572A - Transparent member for holding sample, cover glass, and pinch sample object - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a sample object constitution member capable of constituting the sample object with which the reflection on an undesirable surface of detection light of an autofocusing device is suppressed. <P>SOLUTION: The pinch sample object 110 is comprised of slide glass 120, cover glass 130, and a sample 140 held between the slide glass 120 and the cover glass 130. The sample 140 consists of, for example, an observation object 142, and an encapsulating medium 144, such as glycerol, and has a refractive index of approximately 1.3 to 1.55. The slide glass 120 is subjected to a visible light reflection preventive/near IR light reflective coat 124 on a sample holding surface 120a touching a top surface, i.e., the sample 140. The visible light reflection preventive/near IR light reflective coat 124 has low reflectivity to light of a visible region and high reflectivity to light of a near IR region with respect to the sample 140 of the refractive index of approximately 1.3 to 1.55. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a specimen holding transparent member, a cover glass, and a holding specimen for a microscope equipped with an active autofocus device using near infrared light.

  Recently, research on chromosomes and the genes present therein has made rapid progress, and various devices have been developed for observing and examining chromosomes and measuring fluorescence of genes. Unlike the general observation of pathological specimens (tissue sections), the characteristics of examination and photometry used in this field require examination of many specimens in order to obtain one examination result, or with a 100x objective lens. The observation object in the sample is small enough to be observed at last, and scattered on the slide glass, so that it is difficult to focus on the observation object.

  For example, when observing a DNA chip, fluorescence measurement is performed on several tens to several hundreds of samples provided on a slide glass. In addition, when observing chromosome deformation, it is necessary to search all over the slide glass for chromosomes scattered on the slide glass using a high-magnification objective lens.

  Therefore, what is required is a so-called autofocus (AF) apparatus that automatically focuses on a specimen for automating observation (reducing time and reducing fatigue).

  AF devices are roughly classified into an image processing type (passive type) and a reflected light processing type (active type).

  In the image processing type (passive type), for example, an image of a specimen is captured by a CCD, and the position where the contrast of the image is the highest is focused. Since the focus position is determined by image processing, if there is an image, it can be accurately focused. However, on the other hand, high image contrast is essential for accurate focusing. Further, since a so-called hill-climbing movement of the objective lens that searches for the highest contrast position by processing the images before and after focusing is necessary, it takes time to focus.

  In the reflected light processing type (active type), the AF device itself has a light source for focus detection, the sample is irradiated with detection light emitted from this light source, the reflected light from the sample is received, and the amount of reflected light and the reflected spot are moved The focus is detected based on the above. Since the focus can be detected by the analog circuit, the focus detection time is short, and, for example, AF tracking is possible even when the sample moves on the stage. However, the specimen must reflect the detection light. In the latest AF apparatus, an apparatus that reflects detection light by about 1% also functions. Further, if there is a reflective object different from the observation object in the vicinity of the focus, the reflective object may be brought into focus.

  In addition, as the detection light, light having a wavelength different from the wavelength of the light that creates the observation image is used so as not to disturb the image observation. For visible light image observation, near infrared light of about 700 to 850 nm is used as detection light. In the case of ultraviolet light image observation, visible light is used as detection light.

  In addition to the image processing type (passive type) and reflected light processing type (active type), there is also a composite type that projects an index for focusing on the specimen from the AF device and processes the image. Are included in the image processing type (passive type).

  The present invention is applied to a microscope equipped with a reflected light processing type (hereinafter, active tracking type) AF device. Hereinafter, in this specification, the AF device means an active tracking type AF device using near-infrared light unless otherwise specified.

  FIG. 9 shows an example of the AF optical system of the active AF apparatus. The AF optical system 200 includes a light source 202, a collimating lens 204, a knife edge 206, a polarization beam splitter (PBS) 208, an aberration correction lens 210, a lens 212, a λ / 4 plate 214, and a half mirror 216. , Objective lens 218, knife edge 220, lens 222, and photodetector 224.

  The AF apparatus shown in FIG. 9 projects detection light as a spot onto a specimen, and a case where AF functions on an object (glass surface or observation object) having a reflectance of 1% or more as a basic performance will be described. . The depth of focus will be described separately.

  Here, since the AF device basically functions with the amount of reflected light, it may be pointed out that the expression of functioning with reflectance is not correct. However, if the reflectivity of the object is small, it is not the case that a powerful laser should be used. On a surface with extremely low reflectivity, the reflectivity varies greatly depending on the state of the surface, and stable reflected light cannot be obtained, so stable AF is impossible. Therefore, if an appropriate laser power is selected from the characteristics of the optical system and the light receiving element of the AF device, it is better to express whether the AF is stable or not by the reflectance of the object.

  A method of observing a preparation (clamped specimen) with a microscope equipped with an active tracking AF device and its drawbacks will be described taking human chromosome observation as an example.

  FIG. 11 schematically shows how a preparation is observed with an upright microscope equipped with a low-magnification drying objective lens.

  The preparation includes a slide glass 20, a cover glass 30, and a specimen 40 held between them. Both the slide glass 20 and the cover glass 30 are composed of a single glass plate and have a refractive index of about 1.52. Sample 40 includes chromosome 42 and glycerin 44. The chromosome 42 is fixed to the slide glass 20 and enclosed between the cover glass 30 and glycerin 44 to a thickness of about 5 to 20 microns.

  A low-magnification drying objective lens 50 is disposed above the preparation, and a condenser lens 54 is disposed below the preparation. The space between the dry objective lens 50 and the cover glass 30 is filled with air.

  In FIG. 11, before the AF operation, the drying objective lens 50 is disposed at a position (retracted position) where the focus position is sufficiently separated from the preparation. When the AF operation is started, the dry objective lens 50 is lowered or the preparation is raised by a stage (not shown), and an in-focus position detection (focus search) operation is started. Since the upper surface 30a of the cover glass 30 has a reflectance of approximately 4%, the AF apparatus determines that the upper surface 30a of the cover glass 30 is a focus surface, and performs AF tracking mode (continuously pursuing the focus position) from the focus search operation. Mode).

  The reflectivity at the material interface is determined by the refractive index of the material on both sides of the interface. FIG. 13 shows the state of reflection at normal incidence. The reflectance R at the interface between the substance a and the substance b is expressed by the following formula (1).

R = {(nb−na) / (nb + na)} ² (1)
Here, na is the refractive index of the substance a, and nb is the refractive index of the substance b.

Assuming that the substance a is air and the substance b is a cover glass, na, = 1, and nb = 1.52. Therefore, the reflectance R is R = (0.52 / 2.52) ² ≈0 from the equation (1). .04 = 4%
It becomes.

  That is, AF functions on the upper surface 30a of the cover glass 30, but the image is not visible and there is no meaning of AF. Therefore, the AF is turned off once, the dry objective lens 50 is brought closer to the cover glass 30 by the thickness of the cover glass 30 (0.17 mm), and the AF is turned on. At this time, if there is a reflected light amount of 1% or more from the lower surface 30b of the cover glass 30, the AF functions on the lower surface 30b of the cover glass 30 and a 10 × objective lens having a depth of focus of about 15 microns is used. If there is an object to be observed within about 10 microns below the lower surface 30b of the cover glass 30, an image can be seen.

However, the reflectance at the lower surface 30b of the cover glass 30 is calculated by substituting the refractive index 1.5 of glass and the refractive index 1.47 of glycerin into the equation (1).
R = (− 0.03 / 2.97) ² ≈0 = 0%
Thus, AF does not operate on the lower surface 30b of the cover glass 30.

  Therefore, the AF device continues the focus search operation, and then the focus position of the dry system objective lens 50 matches the upper surface 20a of the slide glass 20. Here, similarly to the lower surface 30b of the cover glass 30, no reflected light amount of 1% or more is generated, and AF does not function on the upper surface 20a of the slide glass 20. However, if the chromosome 42 exists under the detection light spot adjacent to the upper surface 20a of the slide glass 20 and there is a reflected light amount of 1% or more, the chromosome 42 is in focus. However, since the chromosomes and the like that are observation objects are small and scattered on the slide glass 20, even if there is a reflected light amount of 1% or more from the chromosome 42, the chromosome 42 moves as the stage moves. AF will stop functioning. That is, the AF enabled state and the AF disabled state occur at a slight interval, which is contrary to “there is a continuous reflecting object” which is the tracking condition of the tracking AF, so that AF does not actually function.

  Further, when the dry system objective lens 50 further approaches the cover glass 30 by about 1 mm and the focus position of the dry system objective lens 50 matches the lower surface 20b of the slide glass 20, the AF functions. However, the image of the chromosome 42 is not visible, and nothing is useful for observation.

  In other words, in an upright microscope equipped with a low-magnification drying objective lens, AF functions on the upper surface 30a of the cover glass 30 and the lower surface 20b of the slide glass 20. In either case, an image of the chromosome 42 is obtained. I can't.

  FIG. 12 schematically shows how a preparation is observed with an erecting microscope equipped with a high-magnification oil immersion objective lens. As shown in FIG. 12, the space between the oil immersion objective lens 52 and the cover glass 30 is filled with immersion oil.

  The same consideration as in the case of the dry objective lens 50 is performed for the oil immersion objective lens 52, and when the refractive index of the immersion oil is calculated as 1.5, the AF functions only on the lower surface 20b of the slide glass 20. Result. Therefore, an image of chromosome 42 cannot be obtained. Further, in many cases, the space between the lower surface 20b of the slide glass 20 and the condenser lens 54 is filled with immersion oil for high-resolution microscopy. In this case, AF does not work for any surface of the slide.

  In order to solve this problem, Japanese Patent Application Laid-Open No. 8-82747 discloses that a glass slide is coated with an alloy of Au and Pb in a thickness of 1 to 20 nm to have a reflectance of 10 to 20%. A slide glass coated with 20 nm is proposed.

  Here, although it is speculative about the coating material, to explain, the reason why gold (Au) is used is that gold has a low reflectivity with respect to light in the visible range, and with respect to infrared light having a wavelength of 600 nm or more. This is probably because the reflectance is high and light absorption is less than that of other metals. However, since gold adheres to the glass and develops the specimen on the gold surface (so-called glue), the gold and lead alloy improves adhesion to the slide glass, and the same development as glass on this coat. It is considered that the silicon oxide (glass itself) is coated with a protective film made of gold and lead, which has a low mechanical strength on the surface, as is the case with general aluminum and silver mirrors. Silicon oxide coating is a technique commonly applied to metal mirrors.

In accordance with Japanese Patent Application Laid-Open No. 8-82747, the present inventors performed a simulation of the reflectance with water on a glass slide coated with gold and coated with SiO ₂ thereon. This slide glass has a reflectance of about 1% for light of 400 nm to 550 nm, 3% for light of 650 nm, and 6% for light of 750 nm, and is used in an AF apparatus using near infrared light. It was confirmed that it was compatible. Also, it was expected that there was little absorption. In practice, lead is also coated, so reflection and absorption are expected to increase. However, in the case of transmitted bright field observation, if the reflectance and absorption are almost the same over the visible range, the appearance of the image will be less affected. There is no. What is necessary is just to think that the ND (neutral density) filter used for light quantity adjustment was used at the time of observation. That is, it can be said that this slide glass is suitable for the purpose.
JP-A-8-82747

  However, in Japanese Patent Application Laid-Open No. 8-82747, the reflectance is 10% to 20% in the near infrared, so that the reflectance described at 400 nm of visible light is about 1/3 and 600 nm is about 2/3. It is considered to be reflectance, and in epi-illumination, the background of the microscopic image becomes bright and the reflectance of red light is high, so the background is colored red. Further, even in transmission observation, a similar image can be formed because light absorption depends on the wavelength in gold.

  The reason why the reflectance should be increased to such an extent that defects such as coloring occur while knowing a good coating for the spectroscope using AF is considered to be derived from the AF depth.

  The embodiment described in JP-A-8-82747 is exactly what is shown in FIG. The malfunction at this time is due to the fact that the reflectance at the upper surface 30a of the cover glass 30 is about 4%. Therefore, if the light quantity at which the AF device functions is set to 5% or more with a margin, the AF device ignores the upper surface 30a of the cover glass 30 and the slide glass 20 having a reflectance of about 6%. The specimen holding surface 20a can be focused. However, with such a setting, when the distance between the specimen holding surface and the objective lens focus surface varies due to the movement of the stage, the AF device can follow the focus, that is, follow the AF. Disappear.

  FIG. 14 is a graph showing the conceptual depth of focus. In the AF optical system 200 of FIG. 9, the photodetector 224 is a two-divided photodetector having two light receiving portions, and FIG. 14 shows the sum of electromotive forces from the two light receiving portions. Here, the relationship between the graphs of the infrared light reflectance of 20% and 5% and the AF operation limit value of 4% is shown, but the distance between ab is the in-focus depth of the reflectance of 20%, and the distance between cd is reflected. The depth of focus is 5%. From FIG. 14, it can be seen that in order for 4% AF to operate, a reflectance of at least twice, preferably at least four times the operating limit value is desired.

  That is, in order for AF to ignore the upper surface 30a of the cover glass 30 having a reflectance of 4%, a reflectance of about 16% is required. The meaning of 10% to 20% in JP-A-8-82747 is considered to be here.

  Therefore, the AF device of the upright microscope cannot focus on the sample only by making the slide glass 20 of the sandwiched sample body reflect infrared light. This is because, when observing a preparation with a cover glass 30 with a dry objective lens, when the IR reflectance of the slide glass is low, the infrared light reflection by the upper surface 30a of the cover glass 30 is made higher than the reflectance of the slide glass. It is necessary to keep it low enough. Of course, there is no problem if the reflectance of the slide glass is sufficiently higher than 1%.

  Further, JP-A-8-82747 discloses an object to be observed by irradiating a specimen with powerful light such as a mercury lamp because the reflectance in visible light is increased by the infrared light reflection coating. In the epifluorescence observation, which observes the fluorescence generated from the light, there is a greater problem. In this observation method, the fluorescence from the observation object is observed using a combination of an excitation filter / dichroic mirror / absorption filter, and the excitation light reflected from the specimen mounting surface and the long wavelength light leaking from the excitation filter Autofluorescence generated from the slide glass is generated as a flare on the image plane. In order to reduce this flare, it is an important measure to reduce reflection from the upper surface 20a of the slide glass 20 having the highest observation illumination light intensity.

  The present invention has been made in consideration of such a situation, and an object of the present invention is to reduce the reflection of the detection light of the AF device on an undesired surface without adversely affecting the observation light. It is to provide a main body and a specimen constituting member that makes it possible to construct such a specimen. The specimen member includes a specimen holding transparent member and a cover glass, and the specimen holding transparent member includes a slide glass, a petri dish, a well plate, and the like.

  The present invention is directed, in part, to a specimen holding transparent member for a microscope equipped with an autofocus device.

  The specimen-holding transparent member according to the present invention has a low reflectance with respect to light in the visible range on a specimen-holding surface in contact with the specimen, with a refractive index of approximately 1.3 to 1.55, and a near red Visible light anti-reflection / near-infrared light reflection coating with high reflectivity for outside light is applied.

  In another transparent member for holding a sample according to the present invention, a neutral density metal reflective coating having a reflectance of 4% to 10% with respect to light from the visible region to the near infrared region is applied to the sample holding surface in contact with the sample. Has been.

  One aspect of the present invention is directed to a cover glass disposed in contact with a specimen held on a specimen holding transparent member. In the cover glass according to the present invention, an antireflection coat having a low reflectance with respect to air from the visible region to the near infrared region is applied to both surfaces.

  The present invention is directed, in part, to a holding specimen for a microscope equipped with an autofocus device.

  The sandwich specimen according to the present invention includes a slide glass, a cover glass, and a specimen held between them. The slide glass has a refractive index of approximately 1.3 to 1 on a specimen holding surface in contact with the specimen. The .55 specimen has a low reflectance for visible light and a high reflectance for near-infrared light. The cover glass is provided with an antireflection coating having a low reflectance with respect to air from the visible region to the near infrared region on both surfaces.

  Another nipping specimen according to the present invention comprises a slide glass, a cover glass, and a specimen held between them, and the slide glass has a specimen holding surface in contact with the specimen from the visible region to the near infrared region. A neutral density metal reflective coating with a reflectance of 4% to 10% is applied to the light, and the cover glass has a low reflectivity for light from the visible range to the near infrared range for air. Anti-reflective coating with a rate is applied on both sides.

  According to the present invention, it is possible to configure a specimen in which reflection of detection light of the AF device on an undesired surface is suppressed without adversely affecting observation.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings.

First Embodiment FIG. 1 shows a pinch specimen according to a first embodiment of the present invention.

  In FIG. 1, the sandwich specimen 110 is a so-called preparation, and includes a slide glass 120, a cover glass 130, and a specimen 140 held between the slide glass 120 and the cover glass 130.

  The specimen 140 includes, for example, an observation object 142 and an encapsulant 144 such as oil such as glycerin, and has a refractive index of approximately 1.3 to 1.55.

  The slide glass 120 is provided with a visible light reflection preventing / near infrared light reflecting coating 124 on the upper surface, that is, the sample holding surface 120 a in contact with the sample 140. The visible light reflection prevention / near infrared light reflection coating 124 has a low reflectance with respect to light in the visible range with respect to the specimen 140 having a refractive index of approximately 1.3 to 1.55, and has a near infrared range. It has a high reflectivity for light.

  The visible light reflection preventing / near infrared light reflection coating 124 is formed of, for example, a dielectric multilayer film. The dielectric multilayer film can be any one of zirconium oxide (ZrO2), aluminum oxide (Al2O3), titanium oxide (TiO2), magnesium fluoride (MgF2), silicon oxide (SiO2), lanthanum oxide (La2O3), and tantalum oxide (Ta2O5). Or a mixture thereof. Since these materials have substantially the same sample developability on the surface as glass, it is not necessary to provide a necessary developability improving film on the gold coat.

  As is well known, a thin film coat using a dielectric material can have various characteristics by utilizing interference of light. In addition, a coating using a dielectric material can reduce light absorption.

  FIG. 2 shows the reflectance characteristics of the visible light antireflection / near infrared light reflection coating 124 formed of a dielectric multilayer film. FIG. 2 shows the reflectance characteristics for water and glycerin that are mainly used as encapsulants. FIG. 2 also shows the reflectance characteristics with respect to air for comparison. As shown in FIG. 2, the visible light antireflection / near infrared light reflection coating 124 has a reflectance of 1% or less for light in the visible region (400 nm to 650 nm) with respect to both water and glycerin. The reflectance for light in the near-infrared region (750 nm to 850 nm) is 8% or more. The reflectance with respect to 780 nm light often used in the AF apparatus is 16% or more. Also, light absorption is almost 0%.

  As shown in FIG. 1, the slide glass 120 is further provided with an antireflection coating 126 on the lower surface 120b opposite to the specimen holding surface 120a. The antireflection coat 126 has a low reflectance with respect to light from the visible range to the near infrared range.

  The cover glass 130 is provided with an antireflection coating 134 on both the upper surface 130a and the lower surface 130b. The antireflection coat 134 has a low reflectance with respect to light from the visible range to the near infrared range with respect to air.

The antireflection coating 126 and the antireflection coating 134 are not limited to this, but may be composed of, for example, an MgF ₂ film. FIG. 3 shows the reflectance characteristics of an antireflection coating composed of an MgF ₂ film. FIG. 3 shows the reflectance characteristics for water, air, and oil. As can be seen from FIG. 3, the reflectance of the antireflection coating is always the highest with respect to air. The antireflection coat has a reflectance of 2.5% or less with respect to light from the visible region to the near infrared region (400 nm to 850 nm) with respect to air.

  Since the cover glass 130 has the antireflection coating 134 on both the upper surface 130a and the lower surface 130b, the cover glass 130 is substantially free from back. Therefore, when placing the cover glass 130 on the specimen 140 held on the slide glass 120, there is no mistake in the front.

Application to an Erecting Microscope (Drying System Objective Lens Type) FIG. 1 also schematically shows a state in which the holding specimen 110 is applied to an erecting microscope equipped with a drying system objective lens. In FIG. 1, the holding specimen 110 is disposed below the drying objective lens 150 of the upright microscope. The dry objective lens 150 is generally a low-magnification objective lens for biological lenses. In the erecting microscope, since the specimen 140 is observed through the cover glass 130, the dry objective lens 150 is designed in consideration of the thickness of the cover glass 130 (generally 0.17 mm).

  Hereinafter, the focus search operation of the AF apparatus in the upright microscope will be described. In the following description, for the sake of easy understanding, the AF apparatus moves the objective lens up and down to focus. The AF apparatus includes the AF optical system shown in FIG. 9, and the AF detection light is near-infrared laser light having a wavelength of 780 nm. Furthermore, the basic AF possible reflectivity of the AF device is 1%, and the light amount setting at the start of the AF operation of the AF device is set to 2.5%. In contrast, the AF operation is not performed. Further, it is assumed that the initial focus position of the dry system objective lens 150 is arranged at a retracted position (point P) above the upper surface 130a of the cover glass 130.

  When the AF device is turned on, the AF device starts a focus search operation, and the drying objective lens 150 is lowered.

First, the focus position of the dry objective lens 150 reaches the upper surface 130 a of the cover glass 130. An MgF ₂ film is provided on the upper surface 130a of the cover glass 130, and the MgF ₂ film has a reflectivity of about 1.9% for light of 780 nm with respect to air as can be seen from FIG. . For this reason, when the focus position of the dry system objective lens 150 is positioned on the upper surface 130a of the cover glass 130, the AF apparatus does not detect the upper surface 130a of the cover glass 130 as the focus position, and lowers the dry system objective lens 150. to continue.

Next, the focus position of the dry objective lens 150 reaches the lower surface 130 b of the cover glass 130. An MgF ₂ film is also provided on the lower surface 130b of the cover glass 130. The MgF ₂ film has a reflectance of almost zero with respect to the specimen 140, and the lower surface 130b of the cover glass 130 is almost non-reflective. Accordingly, the AF apparatus continues to lower the drying objective lens 150 without detecting the lower surface 130b of the cover glass 130 as the focus position.

  Next, the focus position of the dry system objective lens 150 reaches the surface 142 a of the observation object 142. When there is a reflected light amount of 2.5% or more from the surface 142a of the observation object 142, the AF device determines that the surface 142a of the observation object 142 is a focus surface, and thereafter starts an AF tracking operation. When there is no reflected light amount of 2.5% or more from the surface 142a of the observation object 142, the AF device continues to lower the dry objective lens 150.

  When the observation object 142 is a pathological section that is stained and then dried and encapsulated, the reflectance of the pathological section is high, and the AF apparatus enters an AF tracking operation. However, when the observation object 142 is a chromosome or the like, almost no reflected light is generated from the surface of the chromosome, and the AF device continues to descend the dry objective lens 150.

  When there is no reflected light amount of 2.5% or more from the surface 142 a of the observation object 142, the focus position of the dry objective lens 150 then reaches the upper surface 120 a of the slide glass 120. On the upper surface 120a of the slide glass 120, a visible light reflection preventing / near infrared light reflecting coating 124 is provided. The visible light reflection preventing / near infrared light reflection coating 124 has a reflectance of 2.5% or more with respect to light of 780 nm with respect to the specimen 140. Specifically, as shown in FIG. 2, the visible light antireflection / near infrared light reflection coating 124 has a reflectance of about 16% with respect to glycerin 144 as an encapsulant. For this reason, a sufficient amount of reflected light is generated from the upper surface 120 a of the slide glass 120. The AF apparatus determines that the upper surface 120a of the slide glass 120 is a focus surface, and thereafter starts an AF tracking operation.

  During the AF follow-up operation, the focus position of the AF device follows the detection light reflecting surface that occurs with the movement of the stage, that is, the vertical movement of the upper surface 120a of the slide glass 120. Strictly speaking, the focus position of the AF apparatus is not on the observation object 142 but on the upper surface 120 a of the slide glass 120. However, the observation object 142 such as a chromosome has a thickness of 1 micron or less, is in close contact with the slide glass 120, and is usually located within the depth of focus of the dry objective lens 150. Therefore, an image as if the chromosome is in focus is obtained by the imaging optical system of the microscope.

  As can be seen from the above description, according to the holding specimen 110 of the present embodiment, the upper surface of the slide glass 120 is not adversely affected on the focus search operation of the AF device in the upright microscope without adversely affecting the observation light. A sufficient amount of reflected detection light is generated from 120a. For this reason, a favorable AF operation is enabled.

  In the description so far, the wavelength of light in the near infrared region is 750 nm to 850 nm, but this is merely an example. In principle, the wavelength of the observation light and the wavelength of the detection light are different, and the reflectance of the target detection light reflection surface may be 2.5% or more only with respect to the detection light. That is, the detection light may be light having a wavelength of 900 nm. Further, if the wavelength of the observation light is 250 nm to 300 nm, the detection light may be light having a wavelength of 350 nm, for example. Further, the reflectance of 2.5% is merely an example, and an appropriate value of the reflectance of the detection light reflecting surface is determined by the sensitivity of the AF device, and is not limited to 2.5%. That is, if the AF possible reflectance of the AF apparatus is x%, the IR reflectance of the slide glass with respect to the detection light may be (2-4) x% or more.

  Here, supplementary explanation will be given for AF when the specimen 140 is thick or when the observation object 142 is not in close contact with the slide glass 120 and is separated from the upper surface 120a of the slide glass.

  In FIG. 9, the focus position of the AF detection light and the focus position of the objective lens 218 coincide. In FIG. 10, the focus position of the AF detection light is on the upper surface 120 a of the slide glass 120, and the focus position of the objective lens 218 is in the middle of the specimen 140 attached to the slide glass 120.

  In a microscope, objective lenses having different magnifications of 5 to 100 times are switched and used. Objective lenses having different magnifications have different chromatic aberrations with respect to the detection light. The AF optical system 200 of the AF apparatus includes an aberration correction lens 210 for correcting chromatic aberration. The aberration correction lens 210 is moved along the optical axis according to the objective lens to be used, and the focus position of the objective lens 218 is determined. The condensing position of the detection light is matched.

  By using this function, adjustment as shown in FIG. 10 is also possible. That is, by adjusting the position of the aberration correction lens 210, the focus position of the detection light is intentionally shifted from the focus position of the objective lens 218 by a predetermined distance, and the focus position of the detection light is AF-followed by the detection light reflecting surface. Let Thereby, the focus position of the objective lens 218 follows a position away from the detection light reflecting surface by a certain distance. As a result, it is possible to observe the observation object located at a certain distance from the detection light reflecting surface.

  A deviation amount d between the focus position of the objective lens 218 and the condensing position of the detection light is roughly calculated from the movement amount D of the aberration correction lens 210 and the magnification M of the objective lens by the following equation (2).

D = d × M ² (2)
For example, in order to obtain a deviation of 2 microns in a 50 × objective lens (focus depth of about 2 microns), the aberration correction lens 210 is estimated from D = 2 × 10 ⁻³ × 50 ² = 5 (mm). May be moved 5 mm.

  In general, considering the aberration of the near-infrared light of the objective lens, such a function can be used for an amount of 3 to 5 times the focal depth of the objective lens.

  Next, fluorescence observation will be described. In the fluorescence observation, epi-illumination using a mercury lamp or a xenon lamp is used, and a dichroic mirror and an absorption filter are arranged on an optical path through which the AF detection light passes. These mirrors and filters generally have a property of reflecting or absorbing light having a wavelength shorter than a certain wavelength of visible light, and transmit light having a wavelength longer than visible light. Therefore, since light having a wavelength of 780 nm is used as the detection light, the AF function is not hindered. Of course, the mirror and the filter have a certain degree of reflectivity and absorption effect, and the detection light may be attenuated. In this case, this can be dealt with by increasing the output of the detection light.

Application to an Erecting Microscope (Oil-immersion Objective Lens Type) FIG. 4 schematically shows the holding specimen of this embodiment applied to an erecting microscope equipped with an oil immersion objective lens. As shown in FIG. 4, the holding specimen 110 is disposed below the oil immersion objective lens 152 of the upright microscope. The oil immersion objective lens 152 is also designed in consideration of the thickness of the cover glass 130 (generally 0.17 mm), like the dry objective lens 150.

In order to obtain high resolution, immersion oil 153 is filled between the oil immersion objective lens 152 and the cover glass 130. Similarly, an immersion oil 155 is filled between the slide glass 120 and the condenser lens 154. The refractive index of the immersion oil is approximately 1.5, and the reflectance at the interface between the cover glass 130 and the immersion oil 153 is approximately 0 from the equation (1). This can also be seen from the reflectance characteristics of the antireflection coating composed of the MgF ₂ film shown in FIG. The same can be said for the interface between the slide glass 120 and the immersion oil 155.

  The focus search operation of the AF apparatus in an upright microscope equipped with an oil immersion objective lens is exactly the same as that of an upright microscope equipped with a dry system objective lens.

  In an upright microscope equipped with an oil immersion objective lens, the reflectance at the upper surface 130a of the cover glass 130 is almost zero, so that almost no light is reflected from the upper surface 130a of the cover glass 130. Accordingly, the AF operation can be performed better than an upright microscope equipped with a dry objective lens.

  Further, in an upright microscope equipped with an oil immersion objective lens, the reflectance at the interface between the cover glass 130 and the immersion oil 153 is close to zero even when the cover glass 130 is not provided with the antireflection coating 134. For this reason, in the application of the holding specimen 110 to an upright microscope equipped with an oil immersion objective lens, the cover glass 130 may not be provided with the antireflection coating 134.

  In the upright microscope, the antireflection coating 126 on the lower surface 120b of the slide glass 120 does not function at all for the AF operation regardless of whether the objective lens is a dry objective lens or an oil immersion objective lens. For this reason, in the application of the holding specimen 110 to an upright microscope, the slide glass 120 may not have the antireflection coating 126 on the lower surface 120b. However, it is hard to understand which side of the coat is only on one side, so it is better to coat on both sides.

Application to Inverted Microscope FIG. 5 schematically shows the holding specimen of this embodiment with respect to an inverted microscope. As shown in FIG. 5, the holding specimen 110 is disposed above the objective lens 156 of the inverted microscope. In the inverted microscope, since the specimen 140 is observed through the slide glass 120, the objective lens 156 is designed in consideration of the thickness of the slide glass 120 (generally 1 mm).

  Hereinafter, the focus search operation of the AF apparatus in the inverted microscope will be described. In the following description, it is assumed that the AF apparatus moves the objective lens up and down to focus as in the case of the upright microscope. The setting of the AF device is the same as that for the upright microscope. It is assumed that the initial focus position of the objective lens 156 is disposed at a retracted position below the lower surface 120b of the slide glass 120.

  When the AF device is turned on, the AF device starts a focus search operation and the objective lens 156 is raised.

  First, the focus position of the objective lens 156 reaches the lower surface 120 b of the slide glass 120. An antireflection coating 126 is provided on the lower surface 120 b of the slide glass 120. For this reason, when the focus position of the objective lens 156 is positioned on the upper surface 130a of the cover glass 130, the AF device continues to raise the objective lens 156 without detecting the upper surface 130a of the cover glass 130 as the focus position.

  Next, the focus position of the objective lens 156 reaches the upper surface 120 a of the slide glass 120. On the upper surface 120a of the slide glass 120, a visible light reflection preventing / near infrared light reflecting coating 124 is provided. For this reason, a sufficient amount of reflected light is generated from the upper surface 120 a of the slide glass 120. The AF apparatus determines that the upper surface 120a of the slide glass 120 is a focus surface, and thereafter starts an AF tracking operation.

  According to the sandwiched specimen 110 of the present embodiment, a sufficient amount of detection light from the upper surface 120a of the slide glass 120 can be obtained without adversely affecting the observation light even in the focus search operation of the AF apparatus in the inverted microscope. Reflected light is generated. For this reason, a favorable AF operation is enabled.

  In the focus search operation, when the focus position of the objective lens 156 is arranged in the vicinity of the observation object 142 in advance, the first focus position is ignored, and AF is applied to the second focus position. In the case where the anti-reflection coating 126 is not applied to the lower surface 120b of the slide glass 120, the lower surface 120b of the slide glass 120 may not be provided.

  In the focus search operation described above, the antireflection coating 134 of the cover glass 130 does not function at all. Therefore, even if the cover glass 130 is not provided with the antireflection coating 134, there is no problem. However, for the reason described below, the cover glass 130 is preferably provided with an antireflection coating 134.

  Since the cover glass 130 is generally as thin as 0.17 mm, the focus position of the objective lens 156 is higher than that of the cover glass 130 particularly when the focus position of the objective lens 156 is arranged near the observation object 142 in advance. It is possible that the AF is turned on in a state of being located at. At this time, if the antireflection coating 134 is not applied to the cover glass 130, AF may be applied to the upper surface 130a of the cover glass 130. However, if the cover glass 130 is provided with the antireflection coating 134, it is possible to prevent such an undesirable operation from occurring.

  In observation with an inverted microscope, the holding specimen 110 is usually arranged with the cover glass 130 facing up, but may be disposed with the cover glass 130 facing down. The AF operation in this case is exactly the same as the AF operation in an upright microscope, except that the top and bottom are reversed. If the antireflection coating 134 is applied to the cover glass 130, it is possible to cope with such an observation method.

  Up to now, the holding specimen 110 in which the specimen 140 is sandwiched and fixed between the slide glass 120 and the cover glass 130 has been described. However, in an inverted microscope, a living observation object is placed on the slide glass and passed through the slide glass. In some cases, observation is performed from below. For such a speculum, a living observation object may be placed directly on the slide glass 120 without using the cover glass 130.

Second Embodiment The present embodiment is directed to an alternative to the visible light antireflection / near infrared light reflection coating 124 provided on the upper surface of the slide glass 120 of the first embodiment. In this embodiment, the material of the reflective coat provided on the upper surface of the slide glass is the same as the first embodiment except that the material is the same. Therefore, in the following description, the drawings used in the first embodiment are referred to. I want.

  The slide glass 120 of the present embodiment is provided with a neutral density metal reflective coat on the upper surface, that is, the specimen holding surface 120a in contact with the specimen 140, instead of the visible light reflection prevention / near infrared light reflection coat 124 of the first embodiment. Has been. Other configurations are the same as those in the first embodiment.

  The metal thin film has no phenomenon of interference in a thin film such as a dielectric, and has the optical properties of the metal itself. In the slide glass disclosed in Japanese Patent Application Laid-Open No. 8-82747, gold is used as a material for the reflective coat. Gold has a reflectivity that is approximately half linear from 600 nm to 400 nm, meets the requirements of low reflectivity for visible light and high reflectivity for infrared light. However, the observation image is colored.

  On the other hand, the neutral density metal reflective coat has a substantially constant reflectance, for example, a reflectance of about 10%, for light from the visible range to the near infrared range (400 nm to 850 nm). Neutral density metal reflective coats also do not significantly change light absorption with wavelength. Therefore, the observation image is not colored.

  The neutral density metal reflective coat may be made of, for example, a thin film of chromium (Cr), nickel (Ni), titanium (Ti), or silver (Ag). Of course, a protective coating such as magnesium oxide or silicon oxide may be applied to materials such as aluminum and silver that have weak mechanical properties and undergo oxidation.

  Since the slide glass 120 of the present embodiment has a reflectance exceeding 4% with respect to light in the visible range, it is unsuitable for fluorescence observation, but the image is colored in general bright-field transmission observation. Therefore, an inexpensive slide glass for AF can be provided.

Third Embodiment This embodiment is directed to a cell culture device applied to an inverted microscope.

  In the microscopic examination using an inverted microscope, the living cells are cultured in a container (cell culture vessel) with at least a part of the bottom surface transparent, the cell culture vessel is placed on the stage of the inverted microscope, and the cells in the cell culture vessel are placed from below. I often observe.

  FIG. 6 shows a cross section of a petri dish that is one of the cell culture devices according to the third embodiment of the present invention. FIG. 7 is an exploded perspective view of the petri dish shown in FIG.

  The petri dish 160 is composed of a lower substrate 162 and a cylindrical body 168. The lower substrate 162 and the cylindrical body 168 are fixed by adhesion. The cylindrical body 168 is made of, for example, a ring made of glass, resin, or metal. The lower substrate 162 is a glass plate, and an upper surface is provided with a visible light reflection preventing / near infrared light reflecting coating 164 and a lower surface is provided with an antireflection coating 166. The configuration of the visible light reflection prevention / near infrared light reflection coat 164 is the same as that of the visible light reflection prevention / near infrared light reflection coat 124 of the first embodiment. The configuration of the antireflection coating 166 is the same as that of the antireflection coating 126 of the first embodiment.

  The AF operation for the petri dish 160 of the present embodiment is exactly the same as when the sandwich specimen of the first embodiment is applied to an inverted microscope.

  According to the petri dish 160 of the present embodiment, a sufficient amount of light can be detected from the bottom of the concave portion of the petri dish 160, that is, the upper surface of the lower substrate 162 without adversely affecting the observation light even in the focus search operation of the AF apparatus in the inverted microscope. Reflected light is generated. For this reason, a favorable AF operation is enabled.

  The petri dish 160 of the present embodiment has a structure in which the lower substrate 162 and the cylindrical body 168 are joined, but may have a configuration in which a near infrared light reflecting coat is applied to the inner bottom surface of a normal petri dish. However, since the bottom of a normal petri dish has poor surface accuracy of glass, a structure in which the lower substrate 162 and the cylindrical body 168 are joined as in this embodiment is preferable.

  FIG. 8 is an exploded perspective view of a well plate which is another cell culture device according to the third embodiment of the present invention.

  The well plate 180 includes a lower substrate 182 and an upper substrate 188. The lower substrate 182 and the upper substrate 188 are fixed by adhesion. The upper substrate 188 is made of, for example, a glass, resin, or metal plate, and has a plurality of through holes that define wells. The lower substrate 182 is a glass plate, and an upper surface is provided with a visible light reflection prevention / near infrared light reflection coating 184 and a lower surface is provided with an antireflection coating 186. The configuration of the visible light reflection prevention / near infrared light reflection coat 184 is the same as that of the visible light reflection prevention / near infrared light reflection coat 124 of the first embodiment. The configuration of the antireflection coating 186 is the same as that of the antireflection coating 126 of the first embodiment.

  The AF operation for the well plate 180 of the present embodiment is the same as that of the petri dish 160, that is, exactly the same as the case where the holding specimen body of the first embodiment is applied to an inverted microscope.

  According to the well plate 180 of the present embodiment, a sufficient amount of light from the well bottom surface of the well plate 180, that is, the upper surface of the lower substrate 182, without adversely affecting the observation light even in the focus search operation of the AF apparatus in the inverted microscope. The reflected light of the detected light is generated. For this reason, a favorable AF operation is enabled.

The holding | maintenance sample body by 1st embodiment of this invention is shown typically the state applied with respect to the upright microscope provided with the dry system objective lens. The reflectance characteristics of a visible light anti-reflection / near-infrared light reflecting coating composed of a dielectric multilayer film are shown. The reflectance characteristic of the antireflection coating composed of the MgF ₂ film is shown. 1 schematically shows a holding specimen body of a first embodiment applied to an upright microscope equipped with an oil immersion objective lens. The holding specimen body of this embodiment is typically shown with respect to the inverted microscope. The cross section of the petri dish which is one of the cell culture devices by 3rd embodiment of this invention is shown. FIG. 7 is an exploded perspective view of the petri dish shown in FIG. 6. It is a disassembled perspective view of the well plate which is another cell culture device by 3rd embodiment of this invention. 2 shows an example of an AF optical system of an active AF apparatus. In the AF optical system of FIG. 9, the focusing position of the AF detection light and the focus position of the objective lens are shifted. A mode that a preparation is observed with an erecting microscope equipped with a low magnification dry objective lens is schematically shown. A mode that a preparation is observed with the erecting microscope provided with the high magnification oil immersion objective lens is shown typically. The state of reflection at normal incidence is shown. A graph for conceptual depth of focus is shown.

Explanation of symbols

DESCRIPTION OF SYMBOLS 110 ... Nipping specimen, 120 ... Slide glass, 120a ... Upper surface, 120b ... Lower surface, 124 ... Visible light reflection prevention / near-infrared light reflection coating, 126 ... Antireflection coating, 130 ... Cover glass, 130a ... Upper surface, 130b ... bottom surface, 134 ... anti-reflection coating, 140 ... specimen, 142 ... observation object, 142a ... surface, 144 ... encapsulant, 150 ... dry objective lens, 152 ... oil immersion objective lens, 153 ... immersion oil, 154 ... condenser Lens, 155 ... Immersion oil, 156 ... Objective lens, 160 ... Petri dish, 162 ... Lower substrate, 164 ... Visible light reflection prevention / Near infrared light reflection coating, 166 ... Antireflection coating, 168 ... Cylindrical body, 180 ... Well plate , 182 ... Lower substrate, 184 ... Visible light reflection prevention / near infrared light reflection coating, 186 ... Antireflection coating G, 188 ... upper substrate, 200 ... AF optical system, 202 ... light source, 204 ... collimating lens, 206 ... knife edge, 208 ... polarization beam splitter, 210 ... aberration correction lens, 212 ... lens, 214 ... λ / 4 plate, 216: Half mirror, 218: Objective lens, 220: Knife edge, 222: Lens, 224: Photo detector.

Claims (16)

  This is a specimen holding transparent member for microscopes equipped with an autofocus device, and the specimen holding surface in contact with the specimen has a refractive index of about 1.3 to 1.55, which is low for visible light. A transparent member for holding a specimen, which has a reflectance and is provided with a visible-light antireflection / near-infrared light reflecting coating that has a high reflectance with respect to light in the near infrared region.   The specimen holding transparent member according to claim 1, wherein the visible light reflection preventing / near infrared light reflection coating is formed of a dielectric multilayer film. 3. The dielectric multilayer film according to claim 2, wherein the dielectric multilayer film includes zirconium oxide (ZrO ₂ ), aluminum oxide (Al ₂ O ₃ ), titanium oxide (TiO ₂ ), magnesium fluoride (MgF ₂ ), silicon oxide (SiO ₂ ), oxide A transparent member for specimen holding composed of either lanthanum (La ₂ O ₃ ), tantalum oxide (Ta ₂ O ₅ ), or a mixture thereof.   2. The specimen holding transparent member according to claim 1, wherein an antireflection coating having a low reflectance with respect to light from a visible range to a near infrared range is applied to a surface opposite to the specimen holding surface.   2. The specimen holding transparent member according to claim 1, wherein the specimen holding transparent member has a flat plate shape and is a so-called slide glass.   2. The specimen holding transparent member according to claim 1, wherein the specimen holding transparent member has a concave portion for accommodating the specimen and is a so-called petri dish.   2. The specimen holding transparent member according to claim 1, wherein the specimen holding transparent member has a plurality of recesses for accommodating the specimen and is a so-called well plate.   This is a transparent specimen holding member for microscopes equipped with an autofocus device, and has a neutral density of 4% to 10% with respect to light from the visible range to the near infrared range on the specimen holding surface in contact with the specimen. A transparent member for specimen holding, which is provided with a metal reflective coat.   9. The specimen holding transparent member according to claim 8, wherein the metal reflective coat is formed of a thin film of any one of chromium (Cr), nickel (Ni), titanium (Ti), silver (Ag), and aluminum (Al). .   9. The specimen holding transparent member according to claim 8, wherein an antireflection coating having a low reflectance with respect to light from the visible region to the near infrared region is applied to a surface opposite to the specimen holding surface.   9. The specimen holding transparent member according to claim 8, wherein the specimen holding transparent member has a flat plate shape and is a so-called slide glass.   9. The specimen holding transparent member according to claim 8, wherein the specimen holding transparent member has a recess for accommodating the specimen and is a so-called petri dish.   9. The specimen-holding transparent member according to claim 8, wherein the specimen-holding transparent member has a plurality of concave portions that accommodate the specimen and is a so-called well plate.   A cover glass that is placed in contact with the specimen held on the specimen-holding transparent member, and has an anti-reflection coating on both sides that has a low reflectivity for air from the visible to the near infrared. Being a cover glass.   It is a holding specimen for microscopes equipped with an autofocus device. It consists of a slide glass, a cover glass, and a specimen held between them. The slide glass has a refractive index on the specimen holding surface in contact with the specimen. Visible light reflection prevention / near-infrared light that has a low reflectance for light in the visible region and a high reflectance for light in the near-infrared region for specimens of approximately 1.3 to 1.55 A holding specimen in which a reflection coating is applied, and the cover glass is provided with an antireflection coating having a low reflectance with respect to air from the visible region to the near infrared region on both sides.   This is a holding specimen for microscopes equipped with an autofocus device. It consists of a slide glass, a cover glass, and a specimen held between them. The slide glass is visible on the specimen holding surface in contact with the specimen. A neutral density metal reflective coating with a reflectance of 4% to 10% for light from the near infrared region to the near infrared region is applied, and the cover glass is light from the visible region to the near infrared region for air. A sandwich specimen with an anti-reflection coat that has a low reflectivity on both sides.

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