• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
  • 引用文献
  • 法律状态
  • 优先权
    • 专利摘要:
    Use of a material for the manufacture of a coverslip, a sample holder or a cell culture container. The present invention relates to the use of a material comprising a coating capable of reflecting light of a wavelength between 350 nm and 850 nm, corresponding to the electromagnetic spectrum regions of the VIS, IR and UV, and a transparent substrate VIS light, for the manufacture of a coverslip, a sample holder or a cell culture container, particularly a coverslip, a sample holder or a cell culture vessel used in optical microscopy. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2666712A1
    申请号:ES201631277
    申请日:2016-10-03
    公开日:2018-05-07
    发明作者:Pablo Aitor Postigo Resa；Diego MEGIAS VÁZQUEZ
    申请人:Centro Nac De Investigaciones Oncologicas Cnio；Consejo Superior de Investigaciones Cientificas CSIC；Centro Nacional de Investigaciones Oncologicas CNIO；
    IPC主号:
    专利说明:

    Use of a material for the manufacture of a coverslip, a sample holder or a cell culture vessel DESCRIPTION
     5
    The present invention relates to the use of a material comprising a coating capable of reflecting totally or partially and selectively light of wavelengths between 350 nm and 800 nm, corresponding to the regions of the electromagnetic spectrum of VIS, IR and UV, and a VIS light-transparent substrate, for the manufacture of a coverslip, a sample holder or a cell culture vessel, particularly a coverslip, a sample holder or a cell culture vessel that are used in optical microscopy.

    Therefore, the invention could be framed in the field of sample analysis and characterization techniques, particularly by optical microscopy. fifteen

    STATE OF THE TECHNIQUE

    Normally, the automatic focusing of an image in optical microscopy is performed by measuring the light reflected on a transparent slide or cover, when an IR light is used as a source of illumination, where the detection of the reflected light is performed in parallel to the system of illumination. The problem of poor stability / sharpness of the automatically focused image appears, for example, when oil immersion targets are used whose refractive index is similar to that of the coverslip or that of the specimen holder commonly a glass, quartz or plastic material. transparent and, therefore, the light that is reflected is small.

    Different ways to improve the stability / sharpness of the image automatically focused on an optical microscope can be found in the literature.
     30
    For example, US2010033811 describes a microscope autofocus system that corrects the image of a sample along the optical path using a beam splitter.

    US2011017902 describes autofocus system and its operating procedure 35 to achieve greater speed and robustness in sample measurements
    Biological microscopy. The microscope autofocus system is based on correcting the images of the samples along the optical path; The high speed of measurement of the autofocus system is achieved by making a pattern with light on the sample and measuring, with a space detector, at least two reflection patterns of said pattern. 5

    WO2013063096 describes a multifunction autofocus system based on focusing the sample image with the help of a positioning by reflection and corrections of astigmatism and chromatic aberration along the z axis, that is, to correct the images of the samples along of the optical path. 10

    These systems are complicated and expensive, so it is necessary to develop new systems that help improve the stability / sharpness of the images obtained by optical microscopy.
     fifteen
    DESCRIPTION OF THE INVENTION

    The present invention relates to the use of a material comprising a coating capable of reflecting totally or partially and selectively light of 20 wavelengths between 350 nm and 800 nm, corresponding to the regions of the visible electromagnetic spectrum, of the IR and of the UV, and a light-transparent substrate VIS, for the manufacture of a coverslip, a sample holder or a cell culture vessel, particularly a coverslip, a sample holder or a cell culture vessel that are used in optical microscopy. 25

    As the coverslip, sample holder or cell culture vessel of the present invention reflects totally or partially and selectively light of wavelengths between 350 and 800, corresponding to the regions of the visible, IR and UV electromagnetic spectrum, said coating it can reflect the light emitted by the autofocus system 30 of an optical microscope, whereby the stability and sharpness of the automatically focused image is corrected by an autofocus system in an optical microscope.

    It should be noted that this improvement in sharpness, stability and image quality is also obtained when immersion oil targets are used.

    The coverslip of the present invention reflects UV light, that is, it blocks the passage of UV light therethrough. This fact gives it an advantage when analyzing biological samples that deteriorate under UV light.
     5
    The use of a material that is also capable of transmitting the fluorescence of a biological sample, for example, a sample of human tissue, for the manufacture of a coverslip, a sample holder or a cell culture vessel has a special interest in fluorescence optical microscopy .
     10
    Therefore, the first aspect of the present invention relates to the use (from here the use of the invention) of a material comprising a substrate and a coating characterized in that

     said substrate is transparent to visible light, 15

     the index of refraction of the material of said coating is greater than the index of refraction of the material of said substrate,

     said coating is deposited on at least one face of the substrate, 20

     said coating has a thickness of less than 1 µm, and

     said coating is capable of reflecting totally or partially and selectively, light of wavelengths between 350 nm and 800 nm, corresponding to the regions of the electromagnetic spectrum of the visible, IR and UV,

    for the manufacture of a coverslip, a sample holder or a cell culture vessel.
     30
    In the present invention, "cell culture vessel" is understood as that vessel in which a cell culture can be prepared. Examples of such containers are a Petri dish, or a multiwell plate.

    In a preferred embodiment of the present invention, the coating is deposited on both sides of the substrate. In this way, if one face is damaged or dirty, the other could be used.

    In another preferred embodiment of the invention, the coating is a thin sheet 5 of tantalum oxide of thickness between 25 nm and 200 nm. More preferably, the thin sheet of tantalum oxide has a thickness between 75 nm and 125 nm.

    In the present invention, "transparent light transparent substrate" is understood as that substrate formed by a material capable of transmitting light corresponding to a range of wavelengths of the electromagnetic spectrum between 350 nm and 800 nm.

    In another preferred embodiment, the transparent support is selected from a glass, quartz or a polymer composition and a copolymer. fifteen

    Examples of transparent polymers that can be used as a substrate are polydimethylsiloxane (PDMS), polymethylmethacrylate (PMMA), hydroxy ethyl methacrylate (HEMA) or negative photosensitive epoxy resin comprising 8 epoxy groups (SU8).
     twenty
    Examples of transparent copolymers that can be used as substrate are copolymers based on styrene and polymethylpentene (PMP).

    In a preferred embodiment, the coverslip, the sample holder or the cell culture vessel are used in optical microscopy, whereby the cell culture vessel 25 must be of adequate size in order to be located in the viewing area of an optical microscope.

    In another preferred embodiment, the coverslip, the sample holder or the cell culture vessel reflect the light of the autofocus system of an optical microscope and said light of the autofocus system strikes the surface of said coverslip, said sample holder or said culture vessel cellular, on the face that comprises the coating.

    The angle of incidence depends on the numerical aperture of the target. In the present invention, the coverslip, the sample holder or the cell culture vessel reflects the
    light of the autofocus system of an optical microscope and said light of the autofocus system strikes at any angle and with any objective on the surface of said coverslip, said sample holder or said cell culture vessel, on the face comprising the coating.
     5
    In the present invention, it is understood to "automatically focus" the use of an autofocus system of an optical microscope that focuses an image of a microscope by measuring or detecting the reflection of an incident light from a lighting system on a plate comprising a sample or a plate covering a sample, where the measurement or detection of the reflected light 10 is carried out in parallel to the lighting system.

    The term "incident light of the autofocus system" refers to that light emitting an LED light or a laser system that is part of an autofocus system of an optical microscope in the range of the electromagnetic spectrum 15 corresponding to wavelengths λ between 350 nm and 800 nm.

    In the present invention, specifically at the interface between the coating and the substrate, a refractive index change occurs that also offers advantages in optical microscopy systems, in particular in fluorescence microscopy of total internal reflection (in English, Total internal reflection fluorescence microscopy or TIRFM), in inclined thin illumination microscopy (in English Highly inclined thin illumination or HILO), as well as in nanoscopy techniques, that is, nanometric resolution microscopy as the baseline state depletion system (in English Ground state depletion and single-molecule return or GSDIM) or Stimulation emission depletion (STED).

    The change in the index of refraction that occurs at the interface between the coating and the substrate in the present invention allows the optimization of the light path 30 in the TIRFM and HILO microscopes.

    The change in refractive index that occurs at the interface between the coating and the substrate in the present invention results in an improvement in the reflection of the incident light used by the GSDIM and STED 35 nanometer resolution microscopes resulting in a higher resolution.

    Another aspect of the present invention relates to a material (hereinafter the material of the invention) comprising a coating formed by a thin sheet of tantalum oxide between 25 nm and 200 nm thick and a substrate transparent to the visible light, where the coating is deposited on at least one of the faces of said substrate.

    In a preferred embodiment of the material of the invention, the coating is deposited on both sides of said substrate.
     10
    In another preferred embodiment of the material of the invention, the thin sheet of tantalum oxide of the material of the invention has a thickness between 75 nm and 125 nm.

    Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following examples and figures are provided by way of illustration, and are not intended to be limiting of the present invention.
     twenty BRIEF DESCRIPTION OF THE FIGURES

    FIG. 1. Example of routine autofocus and microscope scheme equipped with infrared light focusing system. (a) Example of autofocus routine and (b) Example scheme of microscope equipped with infrared light focusing system. 25

    FIG. 2. Cross section of samples 1 to 5 obtained by optical reflection in a confocal microscope.

    FIG. 3. Light reflection images with or without the use of the coating on 30 dry and immersion lenses.

    FIG. 4. Images acquired with a 20X objective of immersion in oil for 3 markers (blue green and red) that emit along the visible.
     35
    FIG. 5. Light transmission curve of a coating designed to reflect ultraviolet radiation

    EXAMPLES 5

    The invention will now be illustrated by tests carried out by the inventors, which demonstrates the effectiveness of the product of the invention.

    FIG. 1a shows an example of an autofocus routine, where the 10 autofocus system comprises the following elements:

    (a) Target situation with the sample focused
    1 Focus plane
    2 Objective 15
    3 Distance from the coverslip to the focus plane

    (b) Loss of focus plane
    1 Focus position (out of focus)
    2 Coverslips 20

    (c) Correction of objective position and focus
    1 Laser or Focus LED
    2 Target movement
     25
    (d) Focus recovery mechanism
    1 Sample
    2 Distance from the coverslip to the focus plane

    FIG. 1b shows an example scheme of a microscope equipped with an infrared light focusing system comprising the following elements.

     1 Mounting medium
     2 Sample
     3 Interface between sample and coverslip 35
     4 coverslips
     5 Immersion medium (oil, water or air)
     6 Objective
     7 Autofocus system
     8 Infrared light
     9 Led or laser 5
     10 online ccd camera
     11 Microscope camera for imaging

    The samples are composed of glass plates (slides) coated with tantalum oxide and whose refractive index is around 2 and a wide region of the visible spectrum is transparent in 10. The coating is a thin film deposited by sputtering. Each sample has a different thickness.

    5 depositions have been made on 5 glass plates with 5 different thicknesses, which are 100 nm (1), 75 nm (2), 50 nm (3), 25 nm (4) and 200 nm (5), to illustrate the 15 effect of the thickness of the coating on the sharpness of the image observed by optical microscopy.

    Optical transmission measurements have been made in a confocal microscope of each of the 1 to 5 samples prepared. Images 1 to 5 correspond to 20 thicknesses 100 nm, 75 nm, 50 nm, 25 nm and 200 nm, respectively. Image 0 corresponds to an uncoated glass plate.

    FIG. 2 shows a cross-section of samples 1 to 5. FIG. 2a corresponds to the light reflection at 488 nm and FIG. 2b at 633 nm on each of the two faces.

    In samples 1 to 5, an improvement of the autofocus was obtained regardless of the magnification of the lens used with or without immersion oil. 30

    In general, it is desirable that the green light, typical of fluorescence, be reflected with low intensity, so that in this case the best coating thickness corresponds to case number 1 (100 nm thickness).
     35
    FIG. 3 shows images of light reflection with or without the use of the coating on dry and immersion targets, in both cases there is a clear increase in said reflection.

    FIG. 4 shows images acquired with a 20X objective of immersion in oil 5 for 3 markers (blue green and red) that emit along the visible. An improvement of the image quality is observed thanks to the improvement of autofocus.

    FIG. 5 shows the light transmission curve designed to reflect ultraviolet radiation. In FIG. 5 it is observed how the passage of ultraviolet light is blocked. 10
    权利要求:
    Claims (10)
    [1]

    1. Use of a material comprising a substrate and a coating characterized in that
     said substrate is transparent to visible light,
     the index of refraction of the material of said coating is greater than the index of refraction of the material of said substrate,
     said coating is deposited on at least one face of the substrate,
     said coating has a thickness of less than 1 µm, and 10
     said coating is capable of reflecting totally or partially and selectively, light of wavelengths between 350 nm and 800 nm,
    for the manufacture of a coverslip, a sample holder or a cell culture vessel.
     fifteen
    [2]
    2. Use according to claim 1, wherein the coating is deposited on both sides of the substrate.

    [3]
    3. Use according to any of claims 1 or 2, wherein the coating is a thin sheet of tantalum oxide between 25 nm and 200 nm thick. twenty

    [4]
    4. Use according to claim 3, wherein the thin sheet of tantalum oxide is between 75 nm and 125 nm thick.

    [5]
    5. Use according to any one of claims 1 to 4, wherein the support 25 transparent to visible light is selected from a glass, quartz, a polymer composition and a copolymer.

    [6]
    6. Use according to any of claims 1 to 3, wherein the coverslip, the sample holder or the cell culture vessel are used in optical microscopy. 30

    [7]
    7. Use according to claim 6, wherein the coverslip, the specimen holder or the cell culture vessel reflects the light of the autofocus system of an optical microscope and wherein said autofocus system light strikes the surface of said coverslip, said specimen holder or said cell culture vessel 35, on the face comprising the coating.

    [8]
    8. Material comprising a coating formed by a thin sheet of tantalum oxide of thickness between 25 nm and 200 nm and a transparent substrate in visible light, where the coating is deposited on at least one of the faces of said substrate. 5

    [9]
    9. Material according to claim 8, wherein the coating is deposited on both sides of said substrate.

    [10]
    10. Material according to any of claims 8 or 9, wherein the thin sheet 10 of tantalum oxide has a thickness between 75 nm and 125 nm.
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    同族专利:
    公开号 | 公开日
    WO2018065649A1|2018-04-12|
    ES2666712B1|2019-03-28|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    WO2002074055A2|2001-03-19|2002-09-26|Ikonisys, Inc.|Epifluorecence microscope with improved image contrast|
    DE102006027836B4|2006-06-16|2020-02-20|Carl Zeiss Microscopy Gmbh|Microscope with auto focus device|
    JP2009192708A|2008-02-13|2009-08-27|Hoya Corp|Beam splitter, single-lens reflex digital camera using the same, and autofocus video camera|
    JP2016161707A|2015-02-27|2016-09-05|オリンパス株式会社|Reflective mirror and microscope|
    法律状态:
    2019-03-28| FG2A| Definitive protection|Ref document number: 2666712 Country of ref document: ES Kind code of ref document: B1 Effective date: 20190328 |
    2019-09-12| FA2A| Application withdrawn|Effective date: 20190906 |
    优先权:
    申请号 | 申请日 | 专利标题
    ES201631277A|ES2666712B1|2016-10-03|2016-10-03|USE OF A MATERIAL FOR THE MANUFACTURE OF A CUBREOBJETOS, A PORTALUESTRAS OR A CELLULAR CULTURE VESSEL|ES201631277A| ES2666712B1|2016-10-03|2016-10-03|USE OF A MATERIAL FOR THE MANUFACTURE OF A CUBREOBJETOS, A PORTALUESTRAS OR A CELLULAR CULTURE VESSEL|
    PCT/ES2017/070645| WO2018065649A1|2016-10-03|2017-10-03|Use of a material for the production of a cover slip, a sample holder or a cell culture container|
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