WO2017099755A1 - Microscope polarisant à extinction élevée à codage spectral et procédés d'utilisation - Google Patents

Microscope polarisant à extinction élevée à codage spectral et procédés d'utilisation Download PDF

Info

Publication number
WO2017099755A1
WO2017099755A1 PCT/US2015/064750 US2015064750W WO2017099755A1 WO 2017099755 A1 WO2017099755 A1 WO 2017099755A1 US 2015064750 W US2015064750 W US 2015064750W WO 2017099755 A1 WO2017099755 A1 WO 2017099755A1
Authority
WO
WIPO (PCT)
Prior art keywords
retarder
light
specimen
polarizer
polarization
Prior art date
Application number
PCT/US2015/064750
Other languages
English (en)
Inventor
Thomas E. Milner
Biwei YIN
Jeffrey Russel KUHN
Martin Poenie
Original Assignee
Board Of Regents, The University Of Texas System
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Board Of Regents, The University Of Texas System filed Critical Board Of Regents, The University Of Texas System
Priority to PCT/US2015/064750 priority Critical patent/WO2017099755A1/fr
Publication of WO2017099755A1 publication Critical patent/WO2017099755A1/fr

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J4/00—Measuring polarisation of light
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/21—Polarisation-affecting properties
    • G01N21/23—Bi-refringence
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/0092—Polarisation microscopes
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/36—Microscopes arranged for photographic purposes or projection purposes or digital imaging or video purposes including associated control and data processing arrangements
    • G02B21/365—Control or image processing arrangements for digital or video microscopes
    • G02B21/367—Control or image processing arrangements for digital or video microscopes providing an output produced by processing a plurality of individual source images, e.g. image tiling, montage, composite images, depth sectioning, image comparison
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J4/00—Measuring polarisation of light
    • G01J4/04—Polarimeters using electric detection means
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/21—Polarisation-affecting properties
    • G01N2021/216—Polarisation-affecting properties using circular polarised light

Definitions

  • the invention generally relates to polarized light microscopy.
  • Cell structures such as polymers, membranes or vesicles are birefringent. Birefringence can be detected and imaged based on how these structures interact with polarized light. However, to fully characterize these interactions, the polarized light must be modulated to allow different polarization states incident on the sample.
  • Modulated polarization microscopy has the demonstrated ability to image cytoskeletal elements and other structures in living cells. Although these structures may be structurally smaller than the resolution of the microscope imaging system, such structures can be visualized using MPM. To visualize these structures, images must be acquired while the polarization state of the illuminating light is modulated or varied over time or another domain. State of polarized light that interacts with the sample is modified. Image detail is encoded as small changes in intensity as the polarization state is modulated. Detecting changes in image intensity as one modulates the polarization state allows one to determine specific polarimetric signals (e.g. birefringence) of the specimen from the detected signal.
  • specific polarimetric signals e.g. birefringence
  • Procedures to modulate the polarization state have, heretofore, involved devices such as mechanical rotation of polarizers or waveplates (e.g., '12 wave); liquid crystal retarders, or Faraday rotators.
  • Mechanical rotation of polarizers or 'll-wave plates is limited by the mechanical inertia of the element and is a slow process and frequently introduces mechanical vibration and image blurring.
  • mechanical rotation of a single element e.g., a polarizer or '11- waveplate
  • Liquid crystal retarders can provide a complete sampling of the Poincare sphere but they are slow and they provide poor polarization purity (contrast ratios). Poor polarization purity reduces the intensity changes due cellular birefringence and other polarimetric signals. The slow speed of liquid crystal retarders prevents observation of many cellular processes in real time, while the poor polarization purity limits the types of cellular structures that can be observed.
  • Faraday rotators are fast and compatible with high polarization purity. Faraday rotators, however, introduce technical challenges and do not provide a full sampling of the Poincare sphere and thus may not provide sufficient data to isolate linear and circular birefringence or other polarimetric signals.
  • the accurate modulation of the polarization state using Faraday rotators cancan require generating magnetic field lines that are strictly parallel to the axis of the Faraday rotator rod.
  • generating strictly parallel field lines using electromagnets is impossible.
  • the magnets may be designed such that the field lines are nearly parallel to the optical axis, this leads to inhomogeneous rotation over the cross-section of the Faraday rotator rods.
  • Faraday rotator magnets may require water cooling. Heating of the magnets can distort the relationship between the power and the rotation.
  • Thermistors in the magnets provide temperature measurement that may be used to compensate and correct recorded data.
  • None of these devices are capable of providing the necessary modulation of the polarization state of light at a speed sufficiently fast to visualize moving, living specimens.
  • the polarization microscope generally comprises a variable wavelength light source; a first polarizer optically coupled to the variable wavelength light source, wherein the first polarizer transmits incident light in a pure polarization state; a first retarder module optically coupled to the first polarizer; a specimen stage optically coupled to the first retarder module, wherein the specimen stage holds a specimen in the optical pathway of the light received from the first retarder module; a second retarder module optically coupled to the specimen stage, wherein the second retarder module is an opposite-signed retarder with respect to the first retarder module; a second polarizer or analyzer optically coupled to the second retarder module, wherein the second polarizer or analyzer selects an orthogonal polarization state to the first polarizer; an optical capture device optically coupled to the second polarizer or analyzer, wherein the optical capture device captures light passing through the second
  • a method of visualizing a specimen using a polarization microscope comprising: placing the specimen on a specimen stage of a polarization microscope; and obtaining images of the specimen at one or more wavelengths each corresponding to a polarization state incident on the specimen.
  • FIG. 1 depicts a schematic diagram of a polarization microscope
  • FIG. 2 depicts a schematic diagram of a Poincare sphere
  • FIG. 3a depicts a schematic diagram of an alternate embodiment of a polarization microscope
  • FIG. 3b depicts a schematic diagram of an alternative embodiment of a polarization microscope
  • FIG. 3 c is a top view of the first and second retarder modules from FIG. 3b;
  • FIG. 4 is a cross-sectional view of a schematic diagram of one embodiment of the light source
  • FIG. 5a is a birefringence image of a diatom at high magnification
  • FIG. 5b is a birefringence image of the diatom at a low magnification
  • FIG. 6 is a schematic diagram of the microscope optical train
  • Fig. 7a is an image using the polarization microscope showing a BM3.3 T cell attacking a target and individual microtubules;
  • Fig. 7b is an image using the polarization microscope showing microtubules and actin- based stress fibers.
  • Fig. 7c is an image using the polarization microscope showing a small region of a 3T3 fibroblast cell that includes a microtubule (left panel) decorated with numerous small vesicles (right panel).
  • proximal and distal are applied herein to denote specific ends of components of the instrument described herein.
  • a proximal end refers to the end of an instrument nearer to an operator of the instrument when the instrument is being used.
  • a distal end refers to the end of a component further from the operator and extending towards the surgical area of a patient and/or the implant.
  • references to "one embodiment,” “an embodiment,” “example embodiment,” “various embodiments,” etc., may indicate that the embodiment(s) of the invention so described may include a particular feature, structure, or characteristic, but not every embodiment necessarily includes the particular feature, structure, or characteristic. Further, repeated use of the phrase “in one embodiment,” or “in an exemplary embodiment,” do not necessarily refer to the same embodiment, although they may.
  • a polarization microscope includes a variable wavelength light source; a first polarizer that transmits light in a pure polarization state (linear, circular, elliptical, radial, or azimuthal) optically coupled to the variable wavelength light source.
  • the first polarizer is optically coupled to a first retarder (linear, circular or elliptical).
  • a specimen stage is optically coupled to the first retarder, wherein the specimen stage holds a specimen in the optical pathway of the light received from the first retarder.
  • a second retarder having the opposite-sign to the first retarder, is optically coupled to the specimen stage to receive light that passes through the specimen stage.
  • a second polarizer or analyzer is optically coupled to the second retarder, the second polarizer or analyzer being arranged to select an orthogonal state to the first polarizer.
  • An optical capture system is optically coupled to the second polarizer.
  • variable wavelength light source in one embodiment, is capable of producing light having a wavelength from between about 350nm to about 800nm.
  • the first polarizer and/or the second polarizer is a polarizing prism.
  • the first and second retarders are optically coupled with crystal polarized light rotators.
  • the crystal polarized light rotators may be composed of tellurium dioxide or quartz.
  • the polarization microscope comprises a first crystal polarized light rotator and a second crystal polarized light rotator, wherein the first crystal polarized light rotator and the second crystal polarized light rotator are matched crystal rotators. The degree of rotation of the first polarized light rotator and the second polarized light rotator may be based on the wavelength of the incident light.
  • the optical capture device is a charged coupled device or scientific CMOS imager with adequate sensitivity, resolution and speed of image capture.
  • the retarders are a pair of matched rotators (circular retarders) that cause a phase delay between left and right circular polarized light.
  • the retarders are matched elliptical retarders formed by a rotator (circular retarder) and a waveplate (linear retarder) optically coupled to each other.
  • the retarders are elliptical retarders formed by two or more waveplates oriented with respect to each other.
  • a method of visualizing a specimen using a polarization microscope includes: placing the specimen on a specimen stage of a polarization microscope as described above and obtaining images of the specimen at one or more wavelengths. Obtaining images of the specimen may be performed by periodically changing the wavelength of light impinging on the specimen and capturing images of the specimen after each change of wavelength of light. Each change of wavelength may be accomplished in between 1 nanosecond and 10 millisecond, alternatively at least 1 millisecond or longer for bigger aperture ATOF's, since the response time is going to be longer, whereas acquisition of one image can be accomplished in as little as 1 milliseconds.
  • Imaging of the biological specimen may be performed continuously or intermittently as long as a set of images required for characterizing polarization state changes are acquired within a sufficiently short time so that movement of cellular components is small compared to one pixel size on the specimen.
  • a set of images e.g., 2 to 25 images
  • a pulsed light source is employed so that images are captured as with a strobe.
  • the method further comprises modulating the polarization state of the light on the Poincare sphere to produce both azimuthal (about the poles) and longitudinal (about an equatorial axis) movement on the Poincare sphere by altering the wavelength of the light produced by the variable wavelength light source.
  • the method further comprises: adjusting the orientation axis of the first polarizer with respect to the second polarizer (analyzer) so that the first polarizer and the second polarizer (analyzer) are not fully orthogonal; and obtaining images of the specimen while the first polarizer and second polarizer are not fully orthogonal.
  • the method may also include calibrating the polarization microscope by determining the polarization state of the light when the second polarized light rotator and the second waveplate are removed from the optical path.
  • Determining the polarization state of the light may be performed by methods known in the art of light polarization analysis such as rotating the second polarizer (analyzer) through discrete angles and analyzing the data collected by the optical capture device.
  • the polarizers should polarize all wavelengths of light at a nearly equivalent extinction within the operating wavelength range.
  • the term "retarder” refers to an optical element (composed of one or more optical components) is an optical element that provides an optical phase delay ( ⁇ ) between a pair of orthogonal polarization states.
  • the orthogonal states may be linear states oriented at ninety degrees, left- and right-circular polarization states, or orthogonal elliptical states. Other polarization states may be selected or non-null type of measurement.
  • a retarder is specified by either of the two polarization states (sometimes called eigenstates) that propagate through the retarder element without a phase delay and the phase delay ( ⁇ ) between the two orthogonal eigenstates.
  • a waveplate is linear retarder and is specified by two linear orthogonal states that when propagating through the retarder experience a phase delay.
  • the retarder pair in the microscope are configured so that if these two elements were positioned sequentially light would experience no change in the polarization state.
  • An elliptical retarder can be formed, for example, by a sequential combination of a rotator (circular retarder) and a waveplate (linear retarder). Other combinations are known in the art, for example, an elliptical retarder may be formed from two linear retarders that are oriented at 45 degrees with respect to each other.
  • polarizer refers to optical elements that transmit light in a pure polarization state (linear, circular or elliptical) with high extinction for each light wavelength emitted or selected from the light source.
  • polarizer is an optical element that transmits a pure linear polarization state. Polarizers polarize all wavelengths of light equally within the operating wavelength range.
  • Described herein is a new approach to rapidly modulate the polarization state of light on the Poincare sphere to produce both azimuthal (about the poles) and longitudinal (about an equatorial axis) movement on the Poincare sphere.
  • High speed modulation of the polarization state is combined with a relatively high speed imager (100 Hz or faster frame rates) that can capture images corresponding to discrete polarization states of light interacting with the specimen.
  • a relatively high speed imager 100 Hz or faster frame rates
  • the design of the microscope is simple, and utilizes components that maintain high polarization contrast ratios.
  • the design provides, for obtaining null or near null measurements of polarization states.
  • the microscope design can be easily implemented by other laboratories making it useful for a wide range of studies.
  • Polarized light microscopy provides a different mode of imaging with contrast based on intrinsic structure and spatial orientation.
  • Polarized light microscopy has long been used for imaging spindle microtubules based on their birefringence.
  • cellular structures that can be detected with high contrast under polarized light including various filament systems (actin, microtubule, intermediate filaments and collagen), membrane boundaries including those of the plasma membrane, cellular vesicles and various organelles and cellular structures that show crystalline-like organization.
  • Membrane boundaries exhibit edge
  • Contrast in polarized light images arises from changes in phase and/or amplitude of orthogonally polarized states as they travel through the specimen. For example, form- birefringence is exhibited in cells and tissues by various polymers including collagen and the cell cytoskeleton.
  • the electric field of incident light oscillating perpendicular to the fibers (Ej_) induces surface charges that create an induced field (E 0 ) within the fiber.
  • the induced field (E 0 ) anisotropically modifies forward scattered light so that phase and amplitude of ⁇ is altered relative to the electric field component polarized parallel to the fibers (En).
  • the incremental phase retardation (6i) incurred by the perpendicular component (E ⁇ results in slower light transmission and larger refractive index (n s ) than that experienced by light polarized parallel to the fiber axis (En) with refractive index ii f .
  • Incremental phase retardations (6i) accumulate through fibrous structures and the composite phase retardation ( ⁇ ) between components polarized parallel (Ey) and perpendicular (E ) to the fibers after propagating a distance Z is:
  • ⁇ is given in degrees.
  • forward scattered light may have a scattering anisotropy resulting in differential attenuance of light amplitudes. This quantity is given by the form-biattenuance ( ⁇ ) and sometimes called dichroism.).
  • ⁇ the composite relative attenuation ( ⁇ ) between components polarized parallel (Ey) and perpendicular (Ej_) to the fibers after propagating a distance Z is:
  • ⁇ — ⁇ ⁇ ; (Eq 2) [048] where ⁇ is given in degrees. Effect of form-birefringence (An) and form-biattenuance ( ⁇ ) between parallel (Ey) and perpendicular (Ej_) field components may be accounted for, respectively, by real (An) and imaginary ( ⁇ ) parts of the complex differential wavenumber ( ⁇ ):
  • Relative amplitude and phase between perpendicular (Ej_) and parallel (Ey) field components can be expressed mathematically by the complex relative-amplitude (Ej/Ey ). After forward scattered light propagates through a distance (Z), complex relative-amplitude is given by
  • p re is proportional to form-birefringence (An) and Pi m is proportional to form- biattenuance ( ⁇ ).
  • edge birefringence Another type of polarization modification seen in cells is known as edge birefringence, which can be seen at the boundary between dielectric interfaces such as between water and cell membranes.
  • Edge birefringence is an incompletely understood phenomena thought to arise due to interference at boundaries where light from three different paths mix.
  • Various investigators using microscopy have noted that edge birefringence can allow for determination of boundaries with greater accuracy than is obtainable with other types of microscopy. This feature of edge birefringence is consistent with our own observations and is of notable value in experimental studies. Certain crystalline structures such as bone, glycogen granules are intrinsically birefringent.
  • T cell secretory vesicles can strongly modify the polarization state of light and may be an example of intrinsic birefringence.
  • Another type of polarization change is transient birefringence observed during neuronal action potential propagation.
  • circular birefringence is believed to be present in various cellular constituents (e.g. glucose) previous forms of circular birefringence have not provided contrast to observe these sorts of structures.
  • a modulated polarization microscope that changes the incident polarization state by changing the incident wavelength of light on the specimen provides a novel contrast paradigm for microscopy.
  • Some objects in a specimen may modify the polarization state of incident light on the basis of wavelength.
  • the scattering matrix relates the polarization state of scattered light to that of incident light.
  • the scattering-matrix is generally wavelength dependent.
  • an object with little or negligible birefringence in the specimen may scatter incident light polarizations differently with wavelength. This contrast mechanism is distinct from conventional intrinsic and/or form birefringence which may be weakly wavelength dependent.
  • Any suitable camera with suitable speed, resolution, high sensitivity, and low noise may be used to record the images.
  • the camera frame rate also must be proportionally fast.
  • the numerical precision (bits per pixel) and resolution (number of pixels) is also important.
  • the camera should also have low noise and high sensitivity.
  • Exemplary cameras in the contemporary art that may be used include the Hamamatsu Orca Flash 4, the Andor "Zyla” and similar cameras.
  • liquid crystal rotators were not useful for providing images of cytoskeletal elements in living fast moving cells.
  • Faraday rotators could be used to visualize small structures in living cells, but are limited in that they only rotate plane polarized light and thus cannot sample the entire Poincare sphere.
  • Faraday rotators also require input of large amounts of electrical power into the magnets where heating becomes a problem for consistent operation of the instrumentation.
  • High-current Faraday rotators must be water cooled and, even with cooling, one must constantly compensate for temperature changes in Faraday rotating elements.
  • the set of retarders chosen provides polarization states that can include linear, circular, and ellipitical states.
  • Polarization microscope 100 includes a variable wavelength light source 110.
  • Variable wavelength light source 110 is capable of emitting light at multiple wavelengths in a spectral range of about 300 nm to about 1000 nm. In some
  • variable wavelength light source 110 is capable of producing light having a wavelength from between about 350nm to about 800nm.
  • Variable light source 110 may have a fast switching time between spectral emissions. In some embodiments, the switching time is shorter than the blanking interval between successive frames of the camera. In some
  • variable wavelength light source 110 is capable of changing emission wavelengths at a maximum speed of between 1 microsecond/wavelength and 5000 microseconds/wavelength. Variable wavelength light source 110 may be rapidly switched between emission wavelengths and provide sufficient radiant flux incident on the specimen over a narrow band of wavelengths. Examples of variable wavelength light sources that may be used include, but are not limited to: supercontinuum sources; arc lamps; plasma lamps; induction lamps; combination of diode lasers; tunable lasers, superluminescent diodes (SLED), light emitting diodes (LED); Digital Light Projection (DLP) based devices; etc.
  • Variable wavelength light sources that may be used include two types of light sources: 1) light sources that can simultaneously emit a multiplicity of wavelengths combined with a spectrally tunable filter; or 2) discrete emission wavelength emitting light sources that are switched or tuned over time.
  • An important design consideration for the light source is the power spectral density (W/Hz or W/nm).
  • the power spectral density should be sufficiently large to satisfy at least two requirements.
  • the spectral width (nm) of each emission wavelength should be sufficiently narrow so that the polarization state incident on the sample is nearly constant as represented, for example, on the Poincare sphere.
  • the source light must have sufficient power (mW) so that a small polarization change by the specimen can be detected with a signal to noise ratio larger than unity.
  • mW sufficient power
  • Light sources with a power spectral density of about lmW/nm can satisfy these two requirements.
  • a tunable spectral filter e.g., monochromator, Fabry -Perot filter, acousto-optic filter, or filter wheel
  • a tunable spectral filter e.g., monochromator, Fabry -Perot filter, acousto-optic filter, or filter wheel
  • Light sources that simultaneously emit a multiplicity of wavelengths include supercontinuum sources, arc lamps, and light emitting diodes (LEDs).
  • an exemplary embodiment would be a rapidly tunable laser or light source composed of multiple diode laser elements that are combined in for example a multimode optical fiber.
  • the purpose of the multimode fiber is to spatially decorrelate or render light spatially incoherent.
  • An exemplary variable wavelength light source that uses a lamp and tunable filter is the OL490 Agile Light Source from Optronic Laboratories (Orlando, FL).
  • a bright green LED in combination with a tunable spectral filter can serve as a light source.
  • Exemplary supercontinuum sources are manufactured by KT.
  • An acousto-optic filter can be used to rapidly select a narrow band (l-5nm) of spectral emission. Since the light emitted by the supercontinuum source has a high degree of spatial coherence, light may be coupled into a multimode fiber to provide spatially incoherent light incident on the sample.
  • An exemplary light source that uses discrete laser diodes that are combined using dichroic elements is manufactured by Lumencor (Beaverton, OR). Tunable laser sources may also be used.
  • a laser source with a gain media that covers the spectral range of interest, and includes a tunable element in the laser cavity. The various light emission wavelengths are selected by the tunable element in the laser cavity.
  • Light emitted by the tunable laser is coupled into a multimode optical fiber to reduce spatial coherence.
  • a tunable laser e.g., 1000 - 1 lOOnm
  • a non-linear optical interaction e.g., frequency doubling or parametric conversion
  • the non-linear conversion process must be efficient over the desired spectral range of light incident on the specimen.
  • Polarization microscope 100 includes a first polarizer 120 having a first polarization axis.
  • First polarizer 120 is optically coupled to variable wavelength light source 110, and thus functions as the polarizer for the light source.
  • First polarizer 120 receives light from the variable wavelength light source and converts the light into linear polarized light having an orientation equal to the first polarization axis (arbitrarily depicted in FIG. 1).
  • First polarizer 120 may be a polarizing prism whose performance, preferably, does not depend on the wavelength of incoming light. Examples of polarizing prisms include, but are not limited to, Glan-Thompson prisms, Glan-Taylor prisms, and Glan-Foucault prisms.
  • a second polarizer 160 having a second polarization axis (arbitrarily depicted in FIG. 1), functions as the analyzer. Second polarizer 160 is oriented such that the second polarization axis is orthogonal to the first polarization axis. Second polarizer 160 (analyzer) is also a wavelength independent polarizer. Second polarizer 160 may be a polarizing prism. Preferably, first polarizer 120 and second polarizer 160 are matched polarizers having similar construction and optical properties so that their polarimetric extinction is large (e.g., 10 5 - 10 6 ).
  • first polarizer 120 and second polarizer 160 are two light retarders 130, 150 .
  • a first retarder 130 is optically coupled to first polarizer 120.
  • Second retarder 150 is an opposite- signed retarder with respect to the first retarder.
  • the retarders are rotators (a circular retarder) that cause a phase delay between left and right circular polarized light.
  • an optical element composed of a rotator (circular retarder) and a waveplate (linear retarder) may be used as an elliptical retarder.
  • an optical element composed of two waveplates oriented at 45 degrees, with respect to each other may be used as an elliptical retarder.
  • retarders may be crystal polarized light rotators fabricated from quartz or Te0 2 crystals.
  • the crystal polarized light rotators are formed from left and right rotating version of Te0 2 crystals.
  • Crystal polarized light rotators may rotate linearly polarized light independent of the angular orientation of the crystal such that circularly polarized light remains circularly polarized.
  • the angle of rotation ( ⁇ ) by the crystal polarized light rotator about the pole on the Poincare sphere is a function of wavelength (X), thickness (d) and circular birefringence ( ⁇ ( ⁇ )) of the rotator according to the equation below.
  • the first and second crystal polarized light rotators are matched light rotators (equivalent thickness (d) and circular birefringence ( ⁇ ( ⁇ ))) such that the rotation produced by the first rotator is cancelled by the second rotator .
  • Retarders may also be made from levo- and dextrorotatory optically active organic compounds (enantiomers), or enantiomorphs.
  • Retarders may be made from fixed magnet or electromagnetic Faraday rotators.
  • retarders may be made from thin film polymeric coatings or from suitable nanopatterning of optically transparent materials.
  • Polarization microscope 100 also includes a specimen stage 140 optically coupled to first retarder 130.
  • Specimen stage 140 holds a specimen in the optical pathway of the light received from first polarized light rotator.
  • An optical capture system is optically coupled to the second polarizer (analyzer) 160 to capture light passing through the second polarizer.
  • the optical capture system includes a detector and a processor.
  • the detector may be a sufficiently fast, sensitive, and low noise charged coupled device or scientific CMOS camera.
  • a suitable detector is the Orca Flash 4.0 from Hamamatsu Photonics K.K. (Japan).
  • the detector should be capable of capturing up to 100 frames per second at a resolution of up to 2048 x 2048. Higher frame rates and resolution would also be acceptable.
  • first polarizer 120 is oriented horizontally, after passing through second retarder 150, light will be returned to the horizontal polarization state (if the specimen does not modify the birefringence).
  • Light then passes through the second polarizer (analyzer) 160 oriented vertically, which blocks horizontally polarized light that has not been altered due to birefringence of the specimen. This is the principle herein referred to as a "null measurement”.
  • the maximum light intensity produced by a linearly birefringent object is obtained when the plane of linearly polarized light is oriented 45° with respect to the axis of the birefringent object.
  • the wavelength range required to achieve a 90° rotation about the polar axis on the Poincare sphere gets smaller while the variation of the polarization state over each emission wavelength range becomes larger.
  • a user using the configuration shown in FIG. 1, a user can choose a series of preset wavelengths and record an image at each wavelength. In one embodiment, using a switching time on the order of 20 microseconds or slower, a user can easily switch wavelengths between recording each image. Processing the data may be accomplished using a single frequency Fourier filtering algorithm as set forth in Kuhn et al. "Modulated Polarization
  • Calibration may be necessary to keep the illumination intensity constant and to determine the exact rotation angle achieved for a given wavelength.
  • the variable wavelength light source will allow computer control of the illumination intensity. Determination of rotation angle may be done by using a rotating polarizer.
  • the retarder is an optical element that is composed of a rotator (circular retarder) and a waveplate (linear retarder).
  • first waveplate 225 is optically coupled to first polarizer 220 and the first rotator 230.
  • First waveplate 225 receives polarized light from first polarizer 220 and converts incident linearly polarized light into circularly polarized light and elliptically polarized light.
  • First waveplate 225 variously passes linearly, circularly, or elliptically polarized light (depending on the wavelength) to first rotator 230.
  • First rotator 230 changes the orientation of the linearly or elliptically polarized light.
  • a second waveplate 255 is optically coupled to second polarizer 260 and second rotator 250.
  • Second waveplate 255 reverses the changes created by passage through first waveplate 225 by being oriented at 90 degrees to first waveplate 225.
  • Second waveplate 255 receives circular or elliptically polarized light from second polarized light rotator 250 and converts the incident circular polarized light or elliptically polarized light into linearly polarized light.
  • the linearly polarized light is passed to second rotator 250 which then rotates linear polarized light back to its original angle based on the first polarizer.
  • the waveplates are oriented at +45° and -45° with respect to the polarization axis of first polarizer 220.
  • the waveplates may be made from any suitable birefringent material, such as quartz, mica, and polymers.
  • the first and second waveplates are matched waveplates
  • FIG. 3B Another embodiment of the inverted microscope 300 is shown in FIG. 3B.
  • An incoming light source is optically coupled to a First polarizer 310.
  • Incoming light source could be any sufficiently bright light source that can switch between or otherwise deliver a set of desired wavelengths. This could include broadband sources such as from an arc lamp, a set of laser sources or a tunable laser source.
  • the polarizers ideally are of high quality and provide high extinction (10 5 - 10 6 ). They can be selected from crystal polarizers including calcite or a-barium borate, film polarizers, stretched glass polarizers or any device that produces polarized light.
  • a First retarder module 320 receives polarized light from the first polarizer 310, and the First retarder module 320 comprises a first Quartz retarder 322, a Second Quartz retarder 324, and a first 1 ⁇ 4 wave plate 326.
  • the retarder module 320 includes one or more linear retarders and/or polarization rotators that can be made from crystalline materials such as quartz, Te02 or other materials.
  • the first retarder module 320 is shown in Fig. 3C.
  • the first retarder module 320 is operably coupled to a Condenser 330 which receives light exiting the first retarder module 320.
  • the Condenser 330 is a lens is strain free and non-birefringent, in one embodiment.
  • Condenser 330 then sends light to a Specimen 340. After the Specimen 340, light exits to an Objective lens 342. Objective lens is ideally strain free and non-birefringent.
  • the objective lens 342 then sends light to a Second retarder module 350.
  • the Second retarder module 350 comprises a second 1 ⁇ 4 wave plate 352, a third quartz retarder 354, and fourth quartz retarder 356. As shown in Fig.
  • the second retarder module 350 consists of a series of retarders and or retarders that are matched in thickness, orientation, and optical retardation to those of the first retarder module such that first quartz retarder is matched to the fourth quartz retarder 356, the second quartz retarder 324 is matched to the third quartz retarder 354 and the first 1 ⁇ 4 wave plate 326 is matched to second zero order 1 ⁇ 4 wave plate 352.
  • the fast and slow axes of each retarder are opposite to those of the corresponding retarder in the first retarder module 320.
  • the first 1 ⁇ 4 wave plate 326 and the second 1 ⁇ 4 wave plate 352 are zero order retarders.
  • the second polarizer is similar to the first polarizer except that it is rotated either exactly 90 degrees or 90 degrees + or - a small increment relative to the first polarizers.
  • the first retarder is a 4.25 wave at 525 nm wave retarder
  • the second retarder is a 12.75 wave at 525 nm wave retarder
  • the first wave plate is a zero order retarder at 525 nm.
  • the zero order retarders are two retarders fixed together to give a difference of 1/4 wavelength.
  • the polarization rotators (circular retarders) rotate the polarization state about the polar axis of the Poincare sphere (as a function of wavelength) to give polarized light rotated at different angles
  • the effect of the waveplates is to rotate the polarization state about an equatorial axis on the Poincare sphere.
  • the total retardation and thus circularity of the polarization will be a function of wavelength and the thickness of the waveplates.
  • an approximately 0.5 mm thick quartz waveplate that retards 9.25 ⁇ at 500 nm will retard 8.5 ⁇ at 539.6 nm and 1 1.25 ⁇ at 420.9 nm.
  • the optical elements in the microscope depicted in FIG. 3a can be each represented by a Jones matrix (Table 1) and when multiplied appropriately the matrices show the changes in polarization state as light passes through each element of the optical train.
  • the polarization state was examined for several wavelengths with respect to the retardation plates, to give retardations of 0, 22.5 (1/8 ⁇ ), and 45° (1/4 ⁇ ). The results show that, regardless of the wavelength of illumination, after the last retarder, the light is horizontally polarized giving a null measurement.
  • null measurement allows for detection of weak signals without large swings in brightness of the background illumination. If, for example, one were viewing circularly polarized light through a linear polarizer the image would be very bright. On the other hand when viewing weak birefringence between crossed polarizers, the image would be relatively dim. To do both, it is desirable to limit the brightness of the illumination so that the brightest image is on scale. This in turn limits the sensitivity of the camera to the weak signals needed to detect. With a null measurement, the brightness of illumination is limited by the brightness of the weakly birefringent signals.
  • sample positions with non-zero birefringence give a signal intensity (S,) at wavelength t that is proportional to square of phase retardation ( ⁇ ,), where ( ⁇ 0 , ⁇ 0 ) specifies orientation of the sample birefringence axis relative to the polarization state of light incident on the sample at ⁇ 0 and ⁇ , ⁇ are orthogonal transformations on the Poincare sphere that map the polarization state incident on the sample at wavelength t into the reference state at wavelength ⁇ 0 .
  • ⁇ ⁇ 0 2 (sin 2 ( ⁇ ). sin 2 ( ⁇ ) + cos 2 ( ⁇ ))
  • the signal intensity (Si) for each wavelength ( ⁇ ,) is:
  • Equations for the signal intensity (S,) at each wavelength contain terms that should be calibrated, including: the non-null state ( ⁇ ,, ?, ⁇ ) at each wavelength ⁇ and the orthogonal transformations ( ⁇ ; , ⁇ ,) on the Poincare sphere that relate or map the polarization state incident on the sample at wavelength ⁇ into the reference state at wavelength ⁇ .
  • the goal is to determine the input state for light incident on the specimen so the second crystal rotator and the second waveplate will be removed from the path.
  • polarization analyzers use a rotating 1 ⁇ 4 wave plate.
  • the input state of the second polarizer may be determined by rotating the polarizer through discrete angles by mounting it on a motor-driven rotary stage (e.g., a Picometer, Newport AG- PR100, Newport Corporation, Irvine, CA) placed either in the second turret position or before the camera. Images obtained as the polarizer is rotated will used for the pixel -by-pixel determination of ellipticity (tan(Xi, j )) and orientation ( ⁇ ) of the major axis of the polarization ellipse. Other methods known in the art of light polarization may be used for determining the input polarization state of light.
  • a motor-driven rotary stage e.g., a Picometer, Newport AG- PR100, Newport Corporation, Irvine, CA
  • the handedness should be obvious from the input state but it could also be easily determined by repeating the rotating analyzer measurement with an added achromatic ⁇ /4 plate and determining the axis of the resulting linear polarized state.
  • Each of these terms will be calibrated and fixed during operation of the microscope.
  • One useful feature of the graphics cards is that they can be obtained with large amounts of memory for storing image data. These calibration images take into account and ultimately compensate for aberrations in the lenses that alter the polarization state.
  • S R can be increased by using a brighter light source.
  • spectral range of wavelengths needed to obtain all spectral measurements is broad enough to introduce artifacts into the data. These artifacts include differences in resolution, differential scattering, and differences in absorption. Therefore, it is desirable to minimize the wavelength range needed to obtain all spectral (e.g., 7) measurements.
  • Our data show that a spectral bandwidth of 5nm or less for each wavelength is adequate for accurate calculation of specimen birefringence. Larger spectral bandwidths lead to less accuracy. Better accuracy may be achieved by using narrower bandwidths together with light sources with greater power spectral densities.
  • the use of a narrow range of emission wavelengths is preferred when the objective is to provide the added capability of imaging the same specimen by polarized light or by fluorescence.
  • the bright illumination provided by the polarized light source can bleach fluorescent molecules that absorb in the wavelengths used for polarized light imaging.
  • the use of a narrow range of wavelengths for polarized light imaging allows for fluorescence imaging of molecules whose absorption spectrum lies outside the wavelengths used for polarized light imaging.
  • spectrally-encoded high-extinction polarization microscopy provides a number of unique capabilities.
  • Alternative approaches to multiple discrete fibers may be utilized to control the spatial illumination of light incident on the specimen.
  • a DLP chip can be positioned or imaged to the front focal plane of the condenser lens to control the angle of illumination on the specimen.
  • the polarization microscope may have the capability of fluorescence imaging.
  • the optical polarizers, waveplates and rotators are compact and can be rapidly moved in and out of the optical path to allow the introduction of fluorescent imaging optical components (e.g. a dichroic mirror and suitable filter) into the optical pathway.
  • the polarization optical components can fit into the place of a dichroic filter set in microscopes designed to hold multiple dichroic filter sets. This can allow the use of the standard epifluorescence light path in, for example, the Nikon Eclipse Ti inverted microscope.
  • the fluorescence illumination in this case is simply turned on or off through a shutter.
  • a switching mirror e.g., available from Newport Corporation, Irvine CA
  • one light source such as that provided by a series of lasers can be used for polarized light imaging and a second broad band light source (lamp or supercontinuum) can be used for fluorescence.
  • the laser-based source would enter the vertical light or transmitted light path whereas as the broadband source would illuminate the standard epifluorescence light path (typically through the rear of the microscope).
  • Suitable controls such as shutters or power input into the laser would allow for alternating the illumination between the sources.
  • the camera which may be mounted under the microscope in a linear path, may be used to collect both polarized light and fluorescence images. Alternatively two different cameras might be employed by diverting the output from one port of the microscope to another.
  • the light source is depicted in FIG. 4 as a lamp housing 21.
  • the lamp housing 21 comprises a xenon arc lamp 1 that illuminates an ellipsoidal mirror 2 focused through an ultraviolet-blocking window 3 onto an exit slit 4.
  • a xenon arc lamp is a specialized type of gas discharge lamp, an electric light that produces light by passing electricity through ionized xenon gas at high pressure. It produces a bright white light that closely mimics natural sunlight at ⁇ , ⁇ .
  • Xenon arc lamps can be a continuous-output xenon short-arc lamps or a continuous-output xenon long-arc lamps.
  • the infrared wavelengths from the exit slit 4 pass through a flat mirror 5 onto a beam block 6.
  • the beam block 6 may be water cooled or cooled by another type of cooling mechanism, such as a coolant, fan, refrigerant and the like.
  • the flat mirror 5 is adjustable and in one embodiment, the flat mirror 5 is adjustable through a plurality of screws 7 and the ellipsoidal mirror 2 adjusted through a plurality of posts 8 in reference to a plate 9 and an internal frame 10.
  • the arc lamp anode is attached to a cooler 11 and a ceramic insulating plate 12.
  • the arc lamp cathode is connected to a cooler 13.
  • Anode position adjustment screws 14 pass through the air-tight housing 24 to position the arc lamp 1.
  • Anode and cathode high-voltage connections 15 pass through the case to an external igniter 16 and a power supply 17.
  • Anode coolant lines pass through the case to an external anode radiator and water pump 18.
  • Cathode and beam block coolant lines are connected to a separate radiator and a water pump 19.
  • the housing 24 is filled with a gas through gas line 20.
  • the gas may be nitrogen.
  • the microscope optical train 400 diagram is shown in FIG. 6.
  • the optical path at the bottom has been flipped 180 degrees (around the mirror).
  • the light comes from the light source 410 from the right side and then is reflected upwards into the microscope.
  • the disclosed polarization microscope may be used to study biological events as they occur.
  • the novel polarization microscope may be used to study cytoskeletal dynamics in living cells.
  • polarized light imaging has facilitated understanding of how T cells function. Helper and cytotoxic T cells function by directed and focused secretion of molecules towards another cell. This is largely accomplished by movement of the microtubule organizing center (MTOC) up to the site of contact between a T cell and its cognate target and focusing of secretory vesicles around the MTOC.
  • MTOC microtubule organizing center
  • modulated polarization microscopy may be used to follow microtubules and the MTOC as well as secretory vesicles in T cells.
  • the MTOC organizes the microtubule cytoskeleton in T cells.
  • cytotoxic T cells for example, the CTL engages a target cell and signaling through the T cell receptor leads to T cell activation. This leads to a dramatic reorganization of surface molecules at the target contact site as well as the underlying cytoskeleton.
  • These rearrangements define what is termed the immunological synapse.
  • the MTOC is drawn up to the synapse. Either before or after MTOC movement, secretory vesicles move along microtubules towards the MTOC where they concentrated.
  • polarized microscopes include visualization of the cytoskeleton, visualization of vesicles, membranes, cell organelles, viral particles and organized protein assemblies such as collagen. All of these structures can now be visualized in real time. Polarized light microscopy also can enable the visualization of molecular interactions. Nanorods should be visible based on their interaction with polarized light. Nanospheres, although invisible as monomers, would become visible when dimerized. As labels for individual proteins or receptors, dimerization might be detected when two labeled proteins come together in a binding reaction or when cross-linked by other means.
  • Gold nanoparticles have tremendous potential as labels because as light scatterers, they are orders of magnitude brighter than fluorophores and they do not bleach. Thus one can easily image individual gold nanoparticles. Furthermore, molecule binding events can shift the emission, typically to longer wavelengths. In addition, using polarized light imaging, there is a difference between spherical and rod-shaped nanoparticles or when two spherical nanoparticles come close together. Rod shaped particles depolarize the illumination and exhibit anisotropy whereas individual spherical nanoparticles do not. However, when two spherical nanoparticles come together, with respect to polarized light they behave light rods and exhibit anisotropy. Thus individual molecules tagged with small nanoparticles may be imaged to determine if they are bound or free.
  • Fig. 7a is an image using the polarization microscope showing a BM3.3 T cell attacking a target.
  • the microtubules are pointed out with arrows.
  • BM3.3 is a cytotoxic T cell line that is shown interacting with an EL4 lymphoma cell.
  • the microtubules and microtubule organizing center (MTOC) are evident (arrows) despite the fact that this is a rapidly moving cell.
  • Fig. 7b is an image using the current microscope showing microtubules 500 and actin- based stress fibers 510 with blue arrows. A stress fiber 510 in the process of disintegrating is shown with a dashed arrow.
  • Fig. 7c is an image using the current microscope showing a small region of a 3T3 fibroblast cell that includes a microtubule 500 decorated with numerous small vesicles in the left panel. Some of these vesicles have diameters on the same order as the microtubule (25 nm, right panel). These vesicles can be seen to move along microtubules.

Abstract

L'invention concerne un microscope polarisant qui utilise un codage spectral de l'état de polarisation pour l'imagerie cellulaire. Le microscope polarisant à codage spectral est à la fois suffisamment rapide pour l'imagerie cellulaire et compatible avec une optique à extinction élevée nécessaire pour imager des structures et des ensembles moléculaires. Le microscope à codage spectral permet à l'état de polarisation de la lumière dirigée vers l'échantillon d'échantillonner des états individuels sur la totalité de la sphère de Poincaré tout en donnant simultanément une mesure nulle de la biréfringence cellulaire observée. Un échantillonnage sur la totalité de la sphère de Poincaré permet au microscope de déterminer un retard de phase d'échantillons dû à la fois à une biréfringence linéaire et circulaire. Le microscope polarisant à codage spectral peut fonctionner dans un état légèrement décalé de zéro qui améliorera le rapport signal/bruit.
PCT/US2015/064750 2015-12-09 2015-12-09 Microscope polarisant à extinction élevée à codage spectral et procédés d'utilisation WO2017099755A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/US2015/064750 WO2017099755A1 (fr) 2015-12-09 2015-12-09 Microscope polarisant à extinction élevée à codage spectral et procédés d'utilisation

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/US2015/064750 WO2017099755A1 (fr) 2015-12-09 2015-12-09 Microscope polarisant à extinction élevée à codage spectral et procédés d'utilisation

Publications (1)

Publication Number Publication Date
WO2017099755A1 true WO2017099755A1 (fr) 2017-06-15

Family

ID=59013995

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2015/064750 WO2017099755A1 (fr) 2015-12-09 2015-12-09 Microscope polarisant à extinction élevée à codage spectral et procédés d'utilisation

Country Status (1)

Country Link
WO (1) WO2017099755A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110657954A (zh) * 2018-06-29 2020-01-07 上海微电子装备（集团）股份有限公司 投影物镜偏振像差测量方法
WO2022002939A1 (fr) * 2020-06-30 2022-01-06 Universiteit Gent Procédé pour produire une paire appariée de filtres polarisants et procédé et appareil pour déterminer la concentration de particules biréfringentes au moyen d'une paire de filtres polarisants
WO2022013448A1 (fr) 2020-07-17 2022-01-20 Universite De Bretagne Occidentale Dispositif de caractérisation polarimétrique de l'anisotropie d'un milieu, et système d'imagerie correspondant
US20220247901A1 (en) * 2019-06-20 2022-08-04 Nitto Denko Corporation Set of optical film for image generation system

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007513342A (ja) * 2003-12-03 2007-05-24 パルプ アンド ペーパー リサーチ インスチチュート オブ カナダ 円偏光法、並びにセルロース系繊維の壁厚及び小繊維の方位決定用の機器
JP2016038528A (ja) * 2014-08-11 2016-03-22 株式会社ニコン 偏光顕微鏡および偏光顕微鏡制御装置、並びに円偏光観察方法

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007513342A (ja) * 2003-12-03 2007-05-24 パルプ アンド ペーパー リサーチ インスチチュート オブ カナダ 円偏光法、並びにセルロース系繊維の壁厚及び小繊維の方位決定用の機器
JP2016038528A (ja) * 2014-08-11 2016-03-22 株式会社ニコン 偏光顕微鏡および偏光顕微鏡制御装置、並びに円偏光観察方法

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110657954A (zh) * 2018-06-29 2020-01-07 上海微电子装备（集团）股份有限公司 投影物镜偏振像差测量方法
CN110657954B (zh) * 2018-06-29 2020-09-22 上海微电子装备（集团）股份有限公司 投影物镜偏振像差测量方法
US20220247901A1 (en) * 2019-06-20 2022-08-04 Nitto Denko Corporation Set of optical film for image generation system
WO2022002939A1 (fr) * 2020-06-30 2022-01-06 Universiteit Gent Procédé pour produire une paire appariée de filtres polarisants et procédé et appareil pour déterminer la concentration de particules biréfringentes au moyen d'une paire de filtres polarisants
WO2022013448A1 (fr) 2020-07-17 2022-01-20 Universite De Bretagne Occidentale Dispositif de caractérisation polarimétrique de l'anisotropie d'un milieu, et système d'imagerie correspondant
FR3112605A1 (fr) 2020-07-17 2022-01-21 Université De Bretagne Occidentale - Ubo Dispositif de caractérisation polarimétrique de l’anisotropie d’un milieu, et système d’imagerie correspondant

Similar Documents

Publication Publication Date Title
US20160154229A1 (en) Spectrally-encoded high-extinction polarization microscope and methods of use
US7239388B2 (en) Retardance measurement system and method
Oldenbourg Polarized light microscopy: principles and practice
Mehta et al. Polarized light imaging of birefringence and diattenuation at high resolution and high sensitivity
US8451446B2 (en) Differential polarization measuring extension unit for a laser-scanning microscope
US20040150880A1 (en) Confocal microscope
WO2017099755A1 (fr) Microscope polarisant à extinction élevée à codage spectral et procédés d'utilisation
JP6599352B2 (ja) 遠隔偏光測定装置および方法
Hecht et al. Raman optical activity instrument for studies of biopolymer structure and dynamics
JPH05142141A (ja) 薄膜測定装置
Anastasiadou et al. Experimental validation of the reverse polar decomposition of depolarizing Mueller matrices
CN110715732B (zh) 多功能Stokes-Mueller成像及光谱探测系统和检测方法
JP2002510808A (ja) フーリエスペクトル解析を用いる走査型スポット顕微鏡及びフーリエスペクトル解析の方法
JP3365474B2 (ja) 偏光性イメージング装置
CN116481983A (zh) 一种基于偏振照明的同轴干涉散射显微成像装置及方法
WO2021242855A1 (fr) Procédé et système d'imagerie spectrale de densité, d'anisotropie et d'orientation
WO2019164452A1 (fr) Procédés et appareil de microscopie à fluorescence
US7335874B2 (en) Method and apparatus for generating a circularly polarized illumination beam
Shribak et al. Sensitive measurements of two-dimensional birefringence distributions using near-circularly polarized beam
RU78945U1 (ru) Оптикоэлектронный прибор для определения кальцийсодержащих компонентов строительных растворов
RU2285279C1 (ru) Лазерный сканирующий микроскоп
US1525145A (en) Polarimetric apparatus
Arteaga Barriel et al. Snapshot circular dichroism measurements
WO2023105242A1 (fr) Endoscopie polarimétrique
SU1744688A1 (ru) Устройство дл стереоскопического наблюдени микрообъектов

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 15910376

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 15910376

Country of ref document: EP

Kind code of ref document: A1