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WO2017108136A1 - Liquide d'immersion à indice de réfraction élevé pour imagerie 3d à super-résolution faisant intervenir une optique anail à base de saphir - Google Patents

Liquide d'immersion à indice de réfraction élevé pour imagerie 3d à super-résolution faisant intervenir une optique anail à base de saphir Download PDF

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Publication number
WO2017108136A1
WO2017108136A1 PCT/EP2015/081209 EP2015081209W WO2017108136A1 WO 2017108136 A1 WO2017108136 A1 WO 2017108136A1 EP 2015081209 W EP2015081209 W EP 2015081209W WO 2017108136 A1 WO2017108136 A1 WO 2017108136A1
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WIPO (PCT)
Prior art keywords
immersion
liquid
objective lens
refractive index
immersion liquid
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Ceased
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PCT/EP2015/081209
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English (en)
Inventor
Karen DANIELS
Matthias SCHROETER
Stephan Herminghaus
Junaid LASKAR
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Max Planck Gesellschaft zur Foerderung der Wissenschaften
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Max Planck Gesellschaft zur Foerderung der Wissenschaften
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Priority to PCT/EP2015/081209 priority Critical patent/WO2017108136A1/fr
Publication of WO2017108136A1 publication Critical patent/WO2017108136A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/33—Immersion oils, or microscope systems or objectives for use with immersion fluids
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B26/00—Optical devices or arrangements for the control of light using movable or deformable optical elements
    • G02B26/004—Optical devices or arrangements for the control of light using movable or deformable optical elements based on a displacement or a deformation of a fluid

Definitions

  • the present invention relates to a high refractive index immersion liquid for immersing a front surface of an immersion objective lens of a microscope, and to a combination of such an immersion liquid and an immersion objective lens.
  • the present invention also relates to a kit for preparing such an immersion liquid, and to a method of matching a high refractive index of such an immersion liquid. Further, the present invention relates to a method of using such an immersion liquid. Even further, the present invention relates to a microscope comprising an immersion objective lens, particularly an immersion objective lens of both high numerical aperture and long working distance.
  • the present invention relates to an adaptive lens comprising a cavity delimited by a curved deformable membrane in at least one direction and filled with a high refractive index liquid.
  • JP 2002-053839 A discloses a high refractive index liquid for use in a microscope for enlarging the NA (Numerical Aperture) of an object lens of the microscope.
  • the high refractive index liquid disclosed by J P 2002-053839 A is a liquid mixture of antimony tribromide and an organic compound.
  • the organic compound may be an alcohol, a glycol, an ether, an ester, a ketone, a sulforated hydrocarbon or nitrided hydrocarbon.
  • the mixture consists of 5 parts by weight of diethylene glycol and 334 part by weight of antimony tribromide.
  • the mixture consists of 5 part by weight of diethylene glycol and 50 parts by weight of antimony tribromide.
  • the refractive indices of the two working examples disclosed are reported as being 1 .798 and 1 .831 .
  • Diiodomethane is mentioned as a prior art commercial high refractive index liquid here.
  • a high refractive index liquid is made of diiodomethane containing sulfur.
  • US 2008/0135808 A1 discloses a high refractive index immersion liquid fo r u se i n a fluorescence microscope.
  • the im mersion liq uid comprises d iiodomethane as its main ingredient, and a solid having a high refractive index such as sulfur.
  • a solid having a high refractive index such as sulfur.
  • no other solid of high refractive index to be dissolved in the diiodomethane than sulfur is disclosed.
  • a low auto- fluorescence of the high refractive index liquid is achieved in that its components are purified.
  • This known high refractive index immersion liquid has a reported refractive index of 1 .78.
  • EP 2 466 359 A1 discloses a high refractive immersion liquid for use in a microscope.
  • This immersion liquid comprises an ionic liquid including a metal-halogeno complex anion containing bromine and antimony as a metal element, and a cation.
  • the cation may be an imidazolium cation, a pyridinium cation, a pyrrolidinium cation or an ammonium cation.
  • the immersion liquid may be an 1 : 1 mixture of 1 -butyl-3-methylimidazolium iodide and antimony tribromide.
  • the refractive index of this particular immersion liquid is reported to be 1.80.
  • an immersion medium in the identification of a crystalline material does not involve the problems occurring in using a high refractive index immersion liquid in a microscope particularly in a super-resol ution fl uorescen ce m icroscope i n wh ich low auto-fluorescence and h igh transparency of an immersion liquid are highly relevant.
  • the problem of the invention is solved by a high refractive index immersion liquid according to claim 1 , by a combination of the immersion liquid and an immersion objective lens comprising the features of claim 3, by a kit for preparing the immersion liquid comprising the features of claim 9, by a method of matching a high refractive index of the immersion liquid comprising the features of claim 1 1 , by a method of using the immersion liquid comprising the features of claim 14, by a microscope comprising the features of claim 16, and by an adaptive lens comprising the features of claim 21 .
  • Preferred embodiments of the present invention are defined in dependent claims 2, 4 to 8, 10, 12, 13, 15, 17 to 20, and 22.
  • a high refractive index immersion liquid for immersing a front surface of an immersion objective lens of a microscope comprises a solution of antimony tribromide (SbBrs) dissolved in diiodomethane (CH2I2), wherein the concentration of the antimony tribromide is not more than 50 % by weight.
  • antimony tribromide in the immersion liquid of the present invention At 50 % by weight antimony tribromide in the immersion liquid of the present invention, a saturated solution of the antimony tribromide in the diiodomethane is reached , and first antimony tribromide crystals are formed at room temperature.
  • the antimony tribromide concentration in the high refractive index immersion liquid according to the present invention is below the saturation level of antimony tribromide in diiodomethane to avoid the formation of any light scattering crystals in the immersion liquid.
  • the refractive index of the immersion liquid With a concentration of the antimony tribromide of about 20 % by weight, the refractive index of the immersion liquid will be above 1 .76. A relevant increase in refractive index is already achieved at lower concentrations of the antimony tribromide. Typically, the minimum concentration of the antimony tribromide will be 10 % by weight of the solution.
  • the immersion liquid matches the sapphire in refractive index.
  • the immersion objective lens may particularly be made of sapphire, and it may be a truncated aplanatic immersion objective lens. More particularly, the immersion objective lens may be an aNAIL. Such a combination according to the present invention may both have a high numerical aperture and a high working distance between a front surface of the immersion objective lens and an object or a sample of interest.
  • the combination according to the present invention may comprise a temperature control unit including a controlled temperature surface adjacent to a gap in front of the immersion objective lens to be filled with the index matched immersion liquid.
  • a kit for preparing the immersion liquid according to the present invention includes an amount of antimony tribromide and an amount of diiodomethane.
  • the amount of the antimony tribromide by weight is not more than the amount of the diiodomethane.
  • the amount of the antimony tribromide is not less than a tenth of the amount of the diiodomethane.
  • the kit allows for preparing the immersion liquid of the present invention at various particular refractive indices above 1.74 or above 1.76 and up to 1 .873.
  • a method according to the present invention of matching a high refractive index of the immersion liquid according to the present invention to a high refractive index of a solid medium includes raising the concentration of the antimony tribromide for increasing the refractive index of the immersion liquid and lowering the concentration of the antimony tribromide for decreasing the refractive index of the immersion liquid. Further, the temperature of the immersion liquid may be raised for decreasing the refractive index of the immersion liquid, and lowered for increasing the refractive index of the immersion liquid. Particularly, the concentration of the antimony tribromide may be used for a coarse adjustment of the refractive index of the immersion liquid, whereas the temperature may be used for fine-adjusting the refractive index.
  • the solid medium to which the high refractive index of the immersion liquid is matched according to the present invention may form a front surface of an immersion objective lens or embed a sample of interest.
  • the "solid medium forming the front surface of the immersion objective lens” does not refer to any coating of the immersion objective lens but to the material forming the immersion objective lens as such and determining the basic optical properties of the interface between the immersion objective lens and the immersion liquid.
  • the method according to the present invention of using the immersion liquid according to the present invention in a microscope comprising an immersion objective lens comprises the steps of making the immersion objective lens of sapphire and filling a gap between a front surface of the immersion objective lens and a facing surface of a solid medium including a sample of interest with the immersion liquid matching the sapphire in refractive index.
  • the gap filled with the immersion liquid of sapphire refractive index may be a gap between the immersion objective lens and a facing surface of the sample of interest itself, or the sample of interest may even be floating in the immersion liquid covering the distance between the sample of interest and the immersion objective lens of sapphire.
  • the present invention allows for making immersion objective lenses of sapphire, which are destined for use with immersion liquids of matching refractive index.
  • the refractive index of the immersion liquid according to the present invention depends on the wavelength of the light more strongly than for solid transparent materials of high refractive index like, for example, sapphire, matching the refractive index of the immersion liquid according to the present invention always means matching the refractive index at one particular wavelength.
  • the immersion objective lens of the microscope may be made as a truncated aplanatic immersion objective lens in which the light passing the immersion objective lens is not to be refracted at the front surface but only at the back surface of the immersion objective lens.
  • Such an aplanatic immersion objective lens requires the availability of an immersion liquid precisely matching its refractive index.
  • the present invention provides such an immersion liquid and thus allows for designing a truncated aplanatic immersion objective lens of both a high numerical aperture (NA) and a long working distance (WD).
  • NA numerical aperture
  • WD long working distance
  • the NA may be at least 1.0 or and the WD may be at least 10 mm with a same immersion objective lens.
  • a temperature of the immersion liquid in the gap may be controlled for fine-tuning the refractive index of the immersion liquid.
  • a microscope according to the present invention comprises a sapphire-based immersion objective lens configured to be immersed into an index matched immersion liquid.
  • the immersion objective lens may be a truncated aplanatic immersion objective lens, particularly a so-called aNAIL (aplanatic Numerical Aperture I ncreasing Lens).
  • the truncated aplanatic immersion objective lens may be designed to both have a high numerical aperture (NA) and a long working distance (WD).
  • NA numerical aperture
  • WD long working distance
  • the microscope may comprise a temperature control unit including a controlled temperature surface adjacent to a gap to be filled with the index matched immersion liquid.
  • the controlled temperature surface may be a separate su rface. It may also be the front surface of the immersion objective lens immersed into the index-matches immersion liquid.
  • An adaptive lens according to the present invention comprises a cavity delimited by a curved, deformable and transparent membrane in at least one direction of an optical axis and filled with a high refractive index liquid comprising a solution of antimony tribromide dissolved in diiodomethane, wherein the concentration of the antimony tribromide is at least 10 % and not more than 50 % by weight of the liquid. Due to the high refractive index of the solution of antimony tribromide dissolved in diiodomethane, such an adaptive lens according to the present invention provides for a strong variation in optical properties with comparatively small variations in the curvature of the transparent membrane.
  • the concentration of the antimony tribromide may particularly be in a range from 40 to 50 % by weight, i.e. the concentration may be close to a saturation concentration of the antimony tribromide in diiodomethane.
  • the adaptive lens according to the present invention may comprise a temperature control unit including a controlled temperature surface adjacent to the cavity filled with the high refractive index liquid.
  • the temperature control unit allows for setting a particular temperature of the liquid and thus a particular refractive index of the liquid.
  • the cavity of the adaptive lens according to the present invention may be delimited by transparent membranes in both directions of the optical axis. Alternatively, it may be delimited by the deformable and transparent membrane in one of these directions only, whereas it is delimited by a solid material in the other direction.
  • This solid material may be sapphire-based, and the high refractive index liquid may be composed to match the sapphire in refractive index.
  • Fig. 1 is a schematic of a liquid refractometer setup, based on the design of Nemoto
  • Fig. 2 shows a measurement of laser beam displacement ⁇ by knife-edge scanning.
  • the inset shows the measured power versus the knife position.
  • FIG. 1 is a schematic of the design for simultaneous increase of NA (numerical aperture) and WD (working distance), using sapphire-based aNAIL lens system immersed in the refractive index matching liquid (solution of SbBr3 in CH2I2).
  • aNAI L aplanatic numerical aperture increasing lenses
  • a typical aNAIL design would include a truncated aplanatic solid immersion objective lens of plano-convex shape, made of high refractive index solid material [4-6] such as sapphire [7, 8].
  • a suitable immersion liquid has limited the application of aNAI L to subsurface microscopy of objects or samples immersed inside a refractive index-matched solid medium, without the possibility of depth-scanning [1 , 2, 6].
  • a refractive index-matched immersion liquid will allow for simultaneously harnessing both the high spatial resolution and the depth scanning capability of sapphire-based aNAILs [3].
  • a persistent challenge in the search for high refractive index immersion liquids is to find one with both low absorbance and low scattering.
  • the ideal liquid would provide optical transparency across the full spectrum from ultraviolet to near- infrared, as well as tunability to provide precise index-matching [9].
  • diiodomethane has the key advantage of being commercially available.
  • the strong light scattering and high absorbance of these formulations render them insufficiently transparent for high- resolution optics applications.
  • a lack of knowledge of a salt formulation to increase the refractive index while maintaining optical transparency has caused diiodomethane to remain underutilized as a preferred immersion solvent liquid , despite its inertness with many minerals (including sapphire) [10].
  • the refractive index of an optical medium is typically proportional to its mass density, as described by the Lorentz-Lorentz equation [1 1 ]; this suggests that salts containing heavy elements would be promising candidates.
  • a large electronegativity difference between the salt cation and anion typically predicts improved solubility.
  • the apparatus determines the displacement ⁇ of a laser beam, due to passing through a liquid-filled cuvette rotated by an angle ⁇ with respect to the beam.
  • the center of the laser beam is determined by scanning a knife edge across its profile.
  • the light intensity profile P (x) is well-described by an error function, as expected for a single-mode laser.
  • We identify the center of the beam as the location x at which P (x) rises fastest, as obtained by numerical differentiation.
  • Sample Gaussian beam profiles dP /dx are shown in Fig.
  • the refractive index n of the liquid can be calculated from the following Equation 1 where no is the refractive index of air (the empty cuvette), d is the width of the cuvette, and ⁇ ⁇ - ⁇ is the relative displacement of the Gaussian peak for the liquid-filled cuvette relative to the empty cuvette.
  • Equation 1 already reduces systematic errors due to geometric imperfections of the cuvette by measuring all values of ⁇ against the empty cuvette.
  • Fig. 3 presents the measured values of n as a function of wavelength (panels a-c), temperature (plot abscissa), and concentration (line series).
  • the refractive index can be increased by either changing the concentration (more dissolved salt corresponds to higher n) or the temperature (higher temperature decreases n).
  • preparing a solution of known concentration is more convenient for coarse tuning, and temperature is more convenient for fine tuning in-situ.
  • Optical transparency which can be degraded by both light scattering and absorption, plays a crucial role in determining the utility of an immersion liquid.
  • Fig. 4 shows the transmittance spectra, measured from the near-ultraviolet to the near-infrared.
  • the illumination source is an Ocean Optics tungsten-halogen light source (model HL-2000-FHSA-LL with output power 4.5 mW) and transmitted light is recorded on an Ocean Optics spectrometer (model HR2000+).
  • Ocean Optics spectrometer model HR2000+
  • there are several coincident absorption bands located at ⁇ 725 , 887, and 1037 nm, which possibly arise due to the common solvent CH2I2 used for all five liquids.
  • ⁇ 725 , 887, and 1037 nm, which possibly arise due to the common solvent CH2I2 used for all five liquids.
  • Tyndall effect at all the three wavelengths, with stronger scattering for the Cargille liquids than for our SbBr3 solutions. This suggests that Mie scattering is present, caused by colloidal particles with a size on the same order as ⁇ [18].
  • the beam width also shows a concentration-dependent increase for the SbBr3-CH 2 l 2 liquid solutions. A likely source of particles in this diameter range is that hydrolysis with atmospheric humidity produces small antimony oxide crystals via the reaction 2SbBr 3 + 3H 2 0 ⁇ Sb 2 0 3 + 6HBr [19].
  • Antimony tribromide (SbBrs) dissolved in diiodomethane (CH 2 I 2 ) is a strong candidate as an immersion liquid for sapphire-based aNAI L lenses. Together with a refractive index matched immersion liquid, these lenses allow for the simultaneous increase of both the numerical aperture (NA, the light-gathering power) and the working distance (WD) of an objective lens [2, 3].
  • This technique places the object or sample of interest 27 at the aplanatic point of the spherical surface of the aNAIL 26 in order to have an aberration-free focal spot. It can increase the NA of the backing objective lens by a factor of n 2 a NAii_, up to the maximum achievable value n a NAii_[1 , 2].
  • the lens system depicted was designed using WinLens3D optical design software. Because n is tunable via both concentration and temperature, the liquid formulation presented here could open new routes to creating adaptive lenses with tunable optical power [20]. In these applications, liquid lenses have the advantage of additionally allowing the shape of the lens to be tuned in order to adjust its focal length (and therefore the optical power), mimicking the mechanism of human eye. To date, the lack of an appropriate high-n liquid has limited the range of tunable optical power, as this depends on the difference of the refractive indices of the two immiscible liquids used to build the adaptive liquid lens [21-23].
  • Fig. 6 shows an embodiment of an adaptive liquid lens 1 according to the present invention.
  • the adaptive lens 1 comprises a cavity 2 filled with the high refractive index liquid 3 according to the present invention and delimited by curved membranes 4 in both directions of an optical axis 5.
  • the membranes 4 are both transparent and deformable.
  • the cavity is delimited by a solid lens 7 made of sapphire in one direction of the optical axis 5 and by a membrane 4 in the other direction.
  • the liquid 3 filled in the cavity 2 is refractive index matched to the material of the solid lens 7.
  • a temperature control unit may control the temperature of the solid lens 7 and thus of the liquid 3 contacting the solid lens 7 to ensure the refractive index match . Due to the ch romatic properties of the liquid 3, a full refractive index match will only be achieved at a certain wavelength of the light passing through the adaptive lens 1 along the optical axis 5.

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  • Physics & Mathematics (AREA)
  • Chemical & Material Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Optics & Photonics (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Analytical Chemistry (AREA)
  • Microscoopes, Condenser (AREA)

Abstract

Selon l'invention, un liquide d'immersion à indice de réfraction élevé (29) qui permet d'immerger une surface avant d'un objectif à immersion (26) d'un microscope comprend une solution de tribromure d'antimoine (SbBr3) dissous dans du diiodométhane (CH212). La concentration du tribromure d'antimoine (SbBr3) n'est pas supérieure à 50 % en poids de la solution.
PCT/EP2015/081209 2015-12-23 2015-12-23 Liquide d'immersion à indice de réfraction élevé pour imagerie 3d à super-résolution faisant intervenir une optique anail à base de saphir Ceased WO2017108136A1 (fr)

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PCT/EP2015/081209 WO2017108136A1 (fr) 2015-12-23 2015-12-23 Liquide d'immersion à indice de réfraction élevé pour imagerie 3d à super-résolution faisant intervenir une optique anail à base de saphir

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Cited By (1)

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Publication number Priority date Publication date Assignee Title
CN110826190A (zh) * 2019-10-15 2020-02-21 桂林理工大学 一种基于丁达尔效应溶胶液浓度测量便携式装置的设计方法

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JP2002053839A (ja) 2000-08-08 2002-02-19 Nikon Corp 高屈折率液体
US20060245087A1 (en) * 2003-01-21 2006-11-02 The General Hospital Corporation Microscope objectives
US20080135808A1 (en) 2006-11-29 2008-06-12 Olympus Corporation Immersion oil, method of producing immersion oil, method of reserving immersion oil and reserving vessel for immersion oil
EP2466359A1 (fr) 2010-04-28 2012-06-20 Olympus Medical Systems Corporation Solution d'immersion

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JP2002053839A (ja) 2000-08-08 2002-02-19 Nikon Corp 高屈折率液体
US20060245087A1 (en) * 2003-01-21 2006-11-02 The General Hospital Corporation Microscope objectives
US20080135808A1 (en) 2006-11-29 2008-06-12 Olympus Corporation Immersion oil, method of producing immersion oil, method of reserving immersion oil and reserving vessel for immersion oil
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CN110826190A (zh) * 2019-10-15 2020-02-21 桂林理工大学 一种基于丁达尔效应溶胶液浓度测量便携式装置的设计方法

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