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WO2025157332A1 - Procédé et appareil de détection optique quantitative de la taille, de la position 3d et/ou de l'orientation de diffuseurs de sous-longueur d'onde individuels - Google Patents

Procédé et appareil de détection optique quantitative de la taille, de la position 3d et/ou de l'orientation de diffuseurs de sous-longueur d'onde individuels

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Publication number
WO2025157332A1
WO2025157332A1 PCT/CZ2025/050009 CZ2025050009W WO2025157332A1 WO 2025157332 A1 WO2025157332 A1 WO 2025157332A1 CZ 2025050009 W CZ2025050009 W CZ 2025050009W WO 2025157332 A1 WO2025157332 A1 WO 2025157332A1
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WIPO (PCT)
Prior art keywords
light wave
vortex
detection
optical element
particle
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PCT/CZ2025/050009
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English (en)
Inventor
Marek Piliarik
Milan Vala
Lukasz BUJAK
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Ustav Fotoniky A Elektroniky Av Cr V V I
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Ustav Fotoniky A Elektroniky Av Cr V V I
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Publication of WO2025157332A1 publication Critical patent/WO2025157332A1/fr
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Classifications

    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/06—Means for illuminating specimens
    • G02B21/08—Condensers
    • G02B21/14—Condensers affording illumination for phase-contrast observation
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/002—Scanning microscopes
    • 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
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B27/00—Optical systems or apparatus not provided for by any of the groups G02B1/00 - G02B26/00, G02B30/00
    • G02B27/58—Optics for apodization or superresolution; Optical synthetic aperture systems

Definitions

  • the present invention relates to the field of optical microscopy, in particular, interferometric scattering microscopy, specifically addressing enhanced capabilities in detection of subwavelength particles, such as biomolecules, including improved quantitative measurement and distinction of the amplitude, phase, and/or polarization state of the light scattered from these particles, yielding more precise measurement of the mass, position and/or spatial orientation of the detected subwavelength particles.
  • the invention applies to ultrasensitive microscopy, interferometric scattering microscopy, and mass photometry.
  • optical microscopy Since its inception in the 17 th century, optical microscopy has played a pivotal role in driving significant scientific breakthroughs across the natural sciences. The ability to see the biological matter and its molecular constituents with the resolution limited only by the wavelength of the light itself have enabled ongoing revolution deepening our understanding of the molecular machinery of life. Especially for life sciences, optical microscopy provides an essential means to observe not only the structure, but also the dynamics of the biological systems, from tissues and cells down to single biomolecules, non-invasively and in their native environment.
  • iSCAT interferometric scattering microscopy
  • iSCAT represents a category of techniques that identify and image a subwavelength object by interfering the light scattered on it with a reference light wave.
  • the most common implementation of iSCAT is based on a homodyne common-path interferometer that operates along the optical axis of a microscope, where an interference pattern between the scattered wave and the reference wave is formed. These waves are in general two coherent optical modes of the same frequency but different spatial distributions, yielding an interferogram that is captured by a detector.
  • the scattered wave is much weaker than the reference wave and thus the resulting interferometric contrast is linearly proportional to the scattering amplitude (or polarizability) of the subwavelength object and inversely proportional to the amplitude of the reference wave.
  • the reference wave can be partly attenuated (EP 3923054). Furthermore, the interferometric contrast is modulated by the relative phase between the reference light wave and the scattered wave. Therefore, to achieve a quantitative detection of the scattering amplitude, the relative phase has to be determined as well (Reza Gholami Mahmoodabadi, Richard W. Taylor, Martin Kaller, Susann Spindler, Mahdi Mazaheri, Kiarash Kasaian, and Vahid Sandoghdar. 2020. ‘Point Spread Function in Interferometric Scattering Microscopy (ISCAT). Part I: Aberrations in Defocusing and Axial Localization’. Optics Express 28 (18): 25969-88.
  • phase retrieval has been employed by position scanning or phase scanning of the reference light wave (Hadrien ML Robert, Kristyna Holanova, Lukasz Bujak, Milan Vala, Verena Henrichs, Zdenek Lansky, and Marek Piliarik. 2021. ‘Fast Photothermal Spatial Light Modulation for Quantitative Phase Imaging at the Nanoscale’.
  • the objective of the invention is to provide an improved method of detecting subwavelength particles using iSCAT microscopy, capable of avoiding limitations of conventional techniques and providing independent metrics of the scattering amplitude and the scattering phase and/or the scattering polarization from each detected interferogram.
  • Another objective of the invention is to provide an improved detection apparatus for analyzing samples including subwavelength particles, capable of avoiding limitations of conventional techniques and providing independent metrics of the scattering amplitude and the scattering phase and/or the scattering polarization.
  • the apparatus and method of the present invention are destined for quantitative optical detection of the size, 3D position, and/or orientation of individual subwavelength particles (scatterers) in a sample containing subwavelength particles such as biomolecules and/or molecular ensembles including proteins, protein dimers, protein oligomers, protein complexes, DNA, RNA, lipids, lipid nanoparticles, liposomes, exosomes, virus-like particles, viruses, virus particles, inorganic nanoparticles such as plasmonic or dielectric nanoparticles, and/or nanoparticle oligomers.
  • Virus particles may include adenoviruses and/or lentiviruses and/or retroviruses.
  • subwavelength particles refers to particles having at least one dimension smaller than the wavelength of at least one spectral component of the illumination light wave used to detect them. In a preferred embodiment, the term “subwavelength particles” refers to particles having, in at least one possible conformation of the particle, all dimensions smaller than the wavelength of the illumination light used to detect them.
  • the subwavelength particles have a polarizability corresponding to a molecular mass of 2kDa or more.
  • the apparatus of the present invention is an improved interferometric scattering (iSCAT) microscope that comprises a special optical element, herein called vortex optical element (VOE).
  • VOE vortex optical element
  • Such VOE- based iSCAT microscope may be realized in different configurations, where all of them comprise following components:
  • the detection volume is a three-dimensional space from which the microscope can effectively detect signals from the sample.
  • the detection volume is preferably located within a 100-micrometer distance from the cross-section of the image plane of the microscope objective lens and optical axis, more preferably located within a 10-micrometer distance from the cross-section of the image plane of the microscope objective lens and optical axis.
  • a sample positioning means for holding and/or positioning at least part of the sample within the detection volume. Sample position means may be moved and/or displaced, for example, by a screw- or piezo-driven translation stage.
  • the sample position means typically comprises a means to mount a transparent element comprising a detection surface, wherein the detection surface is a surface of the transparent element, typically a glass coverslip, wherein at least part of the detection surface is located within the detection volume, and there may be a means for receiving the subwavelength particles provided on the detection surface.
  • an illumination source for emitting an illumination light wave to illuminate at least part of the sample located within the detection volume
  • an illumination optical system configured to provide a means to direct and shape the light from the illumination source to illuminate the at least part of the sample within the detection volume
  • a detection optical system configured to collect output light and direct it to the detector; wherein the output light comprises at least part of a scattered light wave, i.e., light scattered on the subwavelength particles to be detected, and at least part of a reference light wave, wherein the reference light wave is light at least partly reflected on the detection surface or at least partly transmitted through the detection volume.
  • the illumination and detection optical system are arranged to enable spatial separation or partial spatial separation of the output light, i.e., light scattered by the subwavelength particles to be detected propagates through the detection optical system as one spatial optical mode and reference light wave propagates through the detection optical system as a second spatial optical mode, and the overlap of the two spatial optical modes is partial or none at a predefined position in the detection optical system, preferably in the Fourier plane.
  • the detector configured to detect the collected output light, said detector is positioned in the detection optical system, preferably in the image plane or at a distance from the image plane, which is smaller than the Rayleigh range of the detection optical system; depending on the configuration of the microscope illumination and detection optical systems, the detector may preferably be a camera with an array of pixels or a point detector such as a photodiode.
  • a vortex optical element positioned in the detection optical system of the microscope, configured to impart different values of a light property (preferably the light property is phase or polarization) to different azimuthal positions of the scattered light wave and to impart a uniform value of the light property to the reference light wave, or configured to impart different values of a light property (preferably the light property is phase or polarization) to different azimuthal positions of the reference light wave and to impart a uniform value of the light property to the scattered light wave.
  • a light property preferably the light property is phase or polarization
  • the vortex optical element is configured to generate a helical wavefront beam or a cylindrical vector beam by at least part of its surface.
  • the vortex optical element can be passive or active.
  • the vortex optical element can be transmissive or reflective.
  • the vortex optical element is located at the predefined position in the detection optical system, preferably in the Fourier plane of the microscope, i.e., a focal plane of the detection optical system that is conjugate to the object plane or image plane, such as a back focal plane of the microscope objective or a secondary Fourier plane realized, for example, using a 4-f system.
  • the present invention further relates to an improved method of detecting subwavelength particles using vortex optical element-based iSCAT apparatus according to the invention.
  • This method is capable of avoiding limitations of conventional iSCAT techniques and provides independent metrics of amplitude and phase and/or polarization of the light scattered from subwavelength particles enabling more precise quantification of the particle size and/or particle mass, particle distance from the detection surface, and/or particle position, and/or particle orientation, and/or particle conformation.
  • the method of the invention comprises the steps of: a) providing a detection surface to at least partly overlap with the detection volume, said detection surface can be positioned within the detection volume using sample positioning means, typically a screw- or piezo-driven translation stages, b) introducing a sample containing subwavelength particles to be detected onto the detection surface, wherein at least part of the sample is within the detection volume; c) emitting illumination light wave from the illumination source, preferably a laser light source, d) directing the illumination light wave into the detection volume by means of the illumination optical system onto the at least part of the sample located within the detection volume, e) causing the illumination light wave to reflect or partially reflect on the detection surface to create a reference light wave, or to transmit or partially transmit through the detection volume to create the reference light wave, f) causing the illumination light wave to scatter in the sample within the detection volume on the subwavelength particles to be detected, thus creating the scattered light wave, g) collecting output light which contains the reference light wave and the scattered light wave by a detection optical system; and causing
  • the information can be obtained by: j) processing and analyzing the interferogram obtained in step i) to yield the scattering amplitude and/or the scattering phase and/or the scattering polarization of each detected subwavelength particle; k) calculating one or more physical properties of the detected subwavelength particles from the scattering amplitude, and/or scattering phase and/or the scattering polarization, based on predefined calibrations, said physical properties being particle size, and/or particle mass, and/or particle distance from the detection surface, and/or particle position, and/or particle orientation, and/or particle conformation; and/or l) calculating at least one focus-independent or position-independent physical property of the detected subwavelength particles.
  • Focus-independent or position-independent physical property may include particle size, and/or particle mass, and/or particle distance from the detection surface, and/or particle position, and/or particle orientation, and/or particle conformation, wherein said calculated physical property is not biased by the measurement uncertainty of the scattering phase of the subwavelength particle due to an uncertainty in its position of the position of the microscope focal plane.
  • the detection surface typically the surface of a clean or functionalized glass coverslip is mounted to the microscope to at least partly overlap with the detection volume.
  • the detection volume is defined by the construction of the illumination optical system and detection optical system.
  • the fine positioning of the detection surface with respect to the microscope detection volume is performed using sample positioning means, typically a screw- or piezo-driven translation stages.
  • the sample containing the subwavelength particles is introduced into the detection volume of the microscope, typically in the form of a liquid sample being in contact with the detection surface of a transparent element such as glass coverslip.
  • Sub wavelength particles may be brought to the detection volume, e.g., by free diffusion or by induced flow. Their detection or dynamic observation may be performed while the particles are immobilized on the detection surface, while the particles are linked at a specific position with respect to the detection surface (e.g. by a chemical tether), while the particles are binding to the detection surface resulting in their immobilization on the detection surface or while the particles are dynamically moving within the detection volume, either freely diffusing, or interacting with various structures that might be attached to the detection surface or at least temporarily present in the detection volume.
  • the structures may include biomolecules, biomolecular ensembles such as membranes, cytoskeletal filaments, cellular organelles, viruses, cells, proteins, protein oligomers, protein complexes, lipid nanoparticles, liposomes, exosomes, virus-like particles, viruses, virus particles, adenoviruses, lentiviruses, retroviruses, antibodies, and other biophysical systems or inorganic nanoparticles such as plasmonic or dielectric nanoparticles, or nanoparticle oligomers.
  • biomolecules such as membranes, cytoskeletal filaments, cellular organelles, viruses, cells, proteins, protein oligomers, protein complexes, lipid nanoparticles, liposomes, exosomes, virus-like particles, viruses, virus particles, adenoviruses, lentiviruses, retroviruses, antibodies, and other biophysical systems or inorganic nanoparticles such as plasmonic or dielectric nanoparticles, or nanoparticle oligomers.
  • the illumination light wave is emitted from the illumination source, typically a laser light source with a spatial and temporal coherence.
  • the illumination light wave is at least partially spatially and temporally coherent.
  • the temporal coherence of the laser should be larger than the distance between the detection surface and the subwavelength particles to be detected, which is typically less than 100 micrometers and the vast majority of available laser light sources fulfill the temporal coherence requirement.
  • the illumination light wave from the illumination source is directed into the detection volume by means of lenses, mirrors, and/or beam-splitters contained within the illumination optical system of the microscope to be partially scattered by the subwavelength particles and partially reflected from the detection surface (in case of reflection arrangement) or partially transmitted through the detection volume (in case of transmission arrangement).
  • the illumination light wave passes through the detection volume and may reflect or partially reflect on the detection surface.
  • the reference light wave is formed from the reflected or partially reflected illumination light wave.
  • the reference light wave is formed from the illumination light wave transmitted through the detection volume.
  • step f) the illumination light wave scatters in the sample within the detection volume on the subwavelength particles to be detected, thus, creating the scattered light wave.
  • the scattered light wave propagates in all directions away from the sample and part of the scattered light wave overlaps spatially with the reference light wave.
  • the output light comprising both scattered light from particles and the reference light wave is collected by the microscope objective, typically an objective with high numerical aperture enabling collection of the light scattered from the particles within wide cone of angles centered along optical axis, and the collected light is then directed using lenses, mirrors, beamsplitters, and other optical elements contained within the detection optical system towards the detector.
  • the microscope objective typically an objective with high numerical aperture enabling collection of the light scattered from the particles within wide cone of angles centered along optical axis
  • the collected light is then directed using lenses, mirrors, beamsplitters, and other optical elements contained within the detection optical system towards the detector.
  • the illumination and detection optical systems are arranged in such a way to enable spatial separation of the output light, i.e., light scattered by particles and reference light wave, into two optical modes with spatially distinct properties.
  • the scattered light wave propagates through the detection optical system as one spatial optical mode and the reference light wave propagates through the detection optical system as a second spatial optical mode.
  • the spatial optical mode of the scattered light wave may differ from the spatial optical mode of the reference light wave in its numerical aperture, direction of propagation field distribution or the beam diameter.
  • the spatial optical mode of the scattered light wave may or may not overlap with the spatial optical mode of the reference light wave.
  • the spatial optical mode of the reference light wave is preferentially by at least 20% smaller in diameter at the position of the vortex optical element than the spatial optical mode of the scattered light wave.
  • both these spatially distinct modes are guided by the detection optical system to be transmitted through or reflected from the vortex optical element being present preferably in one of the Fourier planes of the microscope.
  • the position of the vortex optical element can be displaced along the optical axis of the detection optical system from the exact position of the Fourier plane of the microscope by as much as 50% of the imaging focal length at the cost of decreasing imaging quality.
  • Both light waves transformed by the VOE then propagate towards the detector located near the image plane of the microscope,
  • step i) the interferometric image of the scattered light wave and the reference light wave is detected on the detector, preferably by an image detector, such as a CCD or CMOS camera, or by a point detector, such as a photodiode.
  • an image detector such as a CCD or CMOS camera
  • a point detector such as a photodiode.
  • the digital information of the image is recorded electronically and preferably transferred to the processing electronics of a PC.
  • step j) the resulting interferogram containing interferometric images of the detected subwavelength particles in the form of a vortex interferometric PSF is processed and analyzed to decipher the scattering amplitude and/or the scattering phase and/or the scattering polarization of each detected subwavelength particle.
  • the shape of the vortex interferometric point spread function can be directly compared or fitted with a theoretical model, or it can be compared with an experimental and/or theoretical library of point spread functions, or a deep learning algorithm can be used to interpret the detected point spread function or specific metrics such as the position and contrast of the interferometric maximum and the interferometric minimum can be derived from the experimental data.
  • the specific metric of the point spread function can be directly linked to the scattering crosssection of the detected particle and the phase and/or polarization of the scattered light.
  • the scattering cross-section of the detected particle is independent or very weakly dependent on the particle position and scattering phase of the scattered light.
  • the scattering cross-section of the particle and the phase of the scattered light can be further used to determine the particle mass, the geometrical size of the particle, material properties, axial position, orientation, and/or conformation, denoted as physical properties of detected subwavelength particles in step k).
  • the lateral position of the particle can be directly derived by localizing the position of the point spread function.
  • the physical properties of detected subwavelength particles can be derived directly from the detected interferometric images by fitting a partly informed theoretical model or by deep-learning algorithms trained on theoretical or experimental data.
  • step 1) the resulting interferogram containing interferometric images of the detected subwavelength particles in the form of a vortex interferometric PSF is processed and analyzed to decipher the scattering amplitude independent of the scattering phase and/or the scattering polarization or the scattering phase independent of the scattering amplitude and/or the scattering polarization.
  • the peak-to-peak contrast of the interferogram (determining the scattering amplitude) is independent of the orientation of the interferogram and thus the scattering phase.
  • the physical properties of the detected subwavelength particles linked to the scattering amplitude or the scattering phase or the scattering polarization are calculated as in step k).
  • the focus-independent particle mass is calculated from the peak-to-peak contrast of interferogram independent of the position of the subwavelength particle or the position of the focal plane.
  • the optical pattern generated by the scattered light encoded with the vortex optical element is acquired by a detector and its spatial profile may be interpreted to measure the size of the scattering particles and/or the position of the scattering particles, and/or the orientation of the scattering particles, and/or the material of the scattering particles, and/or the distance between two or more scattering particles.
  • the interference of the scattered light wave with the reference light wave modified by the vortex optical element results in a modified shape of the point spread function in the image plane of the microscope having a specific position of maximum constructive interference and another specific position of a maximum destructive interference, and the contrast and positions of these interference extrema can be used as a quantitative metric to measure the properties of detected subwavelength particles, including their size, molecular weight, 3D position, orientation, or optical anisotropy.
  • Figure 1 A schematic representation of a wide-field implementation of an interferometric scattering microscopy system with epi-illumination and an integrated vortex optical element for vortex interferometric point spread function encoding and phase information retrieval.
  • Figure 2 A schematic representation of a wide-field implementation of an interferometric scattering microscopy system with trans-illumination and an integrated vortex optical element for vortex interferometric point spread function encoding and phase information retrieval.
  • Figure 3 A schematic representation of a scanning confocal implementation of an interferometric scattering microscopy system with epi-illumination, scanning mirror, and an integrated vortex optical element for vortex interferometric point spread function encoding and phase information retrieval.
  • Figure 4 A schematic representation of a scanning confocal implementation of an interferometric scattering microscopy system with epi-illumination, scanning beam deflectors, and an integrated vortex optical element for vortex interferometric point spread function encoding and phase information retrieval.
  • Figure 5 A schematic representation of a scanning confocal implementation of an interferometric scattering microscopy system with confocal illumination, beam scanner, and an integrated vortex optical element for vortex interferometric point spread function encoding and phase information retrieval.
  • Figure 6 A schematic representation of a wide-field implementation of an interferometric scattering microscopy system with epi-illumination and an integrated vortex optical element in the back-focal plane of the microscope objective for vortex interferometric point spread function encoding and phase information retrieval.
  • FIG. 7 Possible realizations of vortex optical element, a) continuous VOE, b) segmented VOE, c) and d) continuous and segmented VOE with a central area having uniform optical properties.
  • VOE is a vortex phase mask
  • VOE is a cylindrical vector beam generator
  • Additional optical element, such as optical filter, lens, or polarizer could be combined with the vortex optical element preferentially within the central area (c,d) primarily to alter the optical properties of the reference light wave.
  • Figure 8 Examples of vortex optical elements changing properties of transmitted light waves, a) a planar wavefront of a light wave transforms into a helical wavefront upon transmission through the vortex phase mask. Tones of grey in the vortex phase mask illustrate the local relative optical thickness of the mask, b) a wave with uniform linear polarization transforms into a cylindrically polarized wave (here radially polarized) upon transmission through a cylindrical vector beam generator, in this case, vortex waveplate. Lines inside the vortex waveplate illustrate the local orientation of the fast axis of the birefringent material.
  • Figure 9 The series of images represents a simulation study demonstrating the use of the novel method under discussion.
  • the image displays the results of placing a point scatterer at varying distances from the focus. It demonstrates how the point scatterer's position impacts the resultant image on the camera.
  • Figure 10 Measured Interferograms of light scattered on silica nanoparticles of a) 80 nm, and b) 120 nm diameter, with a plane reference wave at different axial positions.
  • the Vortex Phase Mask is used to modify the phase profile of the scattered light wave.
  • the z-position of the scattering nanoparticle is denoted on each sub-figure. Scalebar 500 nm.
  • Figure 11 Focus sweep of 20 nm gold nanoparticle, a) experimental images of 20-nm particle at different focus positions (denoted on each sub-figure), b) the spatial cross-section of the interferogram along the angle between the lobes at different focus positions ranging between 0 nm and 550 nm. c) The dependence of the peak-to-peak contrast of the interferogram on the displacement of the focus. Scalebar 500 nm.
  • Figure 12 SEM images of gold nanorods oriented at 45 degrees increments (bottom row) and their respective vortex interferometric point spread functions (top row) measured using an iSCAT microscope with the cylindrical vector beam generator placed near the Fourier plane of the microscope. Scalebar length is 200 nm.
  • Particle size is the geometrical diameter of the subwavelength particle, in the case of an asymmetric subwavelength particle at least the particle size refers to the diameter in at least one direction.
  • Particle mass is the molecular mass of the subwavelength particle or equivalent metric derived from the polarizability of the subwavelength particle.
  • Particle distance from the detection surface is the orthogonal distance between the detection surface and the center of mass of the subwavelength particle.
  • Particle position is the one- two- or three dimensional coordinate defining the position of the center of mass of the subwavelength particle relative to a predefined reference point, the reference point can be a comer of the field of view on the detection surface or another detected particle.
  • Particle orientation is the angle of rotation of the axis of asymmetry of an anisotropic (or non- spherical) subwavelength particle.
  • the particle orientation is derived from the measured anisotropy (polarization dependence) of the scattered light scattered by the subwavelength particle.
  • Particle conformation is shape of the subwavelength particle. Particle conformation refers to a feature of the subwavelength particle in the case, that the subwavelength particle can be found in at least two states, having different asymmetries. Typically, the particle conformation corresponds to protein conformation, lipid vesicle conformation (i.e. collapsed or filled), arrangement of nanoparticle or biomolecular oligomers.
  • Predefined calibrations is a set of experimental or theoretical images of subwavelength particles or parametric descriptions of the images of subwavelength particles which can be used to associate experimental point spread function with physical properties of the subwavelength particle, preferably particle size, particle mass, particle distance from the detection surface, particle position, particle orientation, or particle conformation.
  • Transparent element is an element for carrying the sample, said element comprising a detection surface, wherein the detection surface is a surface of the transparent element, typically glass coverslip, wherein at least part of the detection surface is located within the detection volume, wherein there is a means for receiving the subwavelength particles configured on the detection surface or within a 100- micrometer distance from the detection surface.
  • the means for containing the sample may be provided on the detection surface or instead of the transparent element.
  • the means for containing the sample may typically be a flow channel, a cuvette, a gasket or a surface capable of supporting a droplet of the sample via surface tension.
  • the means for containing the sample is provided instead of the transparent element, it needs to be configured for containing the liquid, leak-proof, and have at least one transparent side with a detection surface.
  • Reference signs used in Figures 1-6 denote: 1 - detection volume, 2 - detection surface, 3- sample positioning means, 4 - subwavelength particle, 5- illumination light wave, 6 - scattered light wave, 7 - reference light wave, 8 - microscope objective, 9, 9a - Fourier plane, 10 - beam splitter, 11, 12, 13,
  • the vortex optical element (VOE) 14 is an optical element with azimuthally dependent optical properties through which the VOE alters the properties of the light wave it interacts with by means of transmission or reflection.
  • the present invention involves two distinct classes of vortex optical elements, herein denoted as vortex phase mask and cylindrical vector beam generator, which differs in the property of light they affect.
  • vortex phase mask when the affected property of the light wave is the phase, we will a use a general term vortex phase mask for all embodiments of such phase-affecting vortex optical elements.
  • vortex phase mask for all embodiments of such phase-affecting vortex optical elements.
  • cylindrical vortex beam generator for all embodiments of polarization-affecting vortex optical elements.
  • Both of these types of vortex optical elements i.e., vortex phase mask and cylindrical vector beam generator, can be realized with a continuously (Figure 7a, c) or discretely (Figure 7b, d) changing optical properties and they may have a singularity in the center, i.e., a point, where all azimuthal components of the optical properties coincide and so the optical properties at that point are not defined ( Figure 7a, b), or may incorporate an area, typically in the central part, within a predefined diameter, or in the outer part, outside of the predefined diameter, having uniform optical properties including free space optical properties such as an open aperture (Figure7c,d).
  • the vortex optical element can be a passive or active optical device, wherein the optical properties of the active vortex optical element may be controlled, for example, by electrical, optical, or thermal means.
  • Vortex phase mask is a vortex optical element that transforms the phase of a light wave transmitted through or reflected from the vortex phase mask, typically by having a non-uniform spatial distribution of optical path length.
  • the vortex phase mask is a transmissive or reflective optical element having azimuthally dependent optical thickness or a surface relief with azimuthally dependent height, respectively.
  • OPL optical path length
  • the vortex phase mask including passive optical elements with azimuthally dependent optical properties such as a transparent window with azimuthally varying thickness, phase mask with varying optical thickness, special subwavelength gratings, metasurfaces, or deformable mirror, or active optical devices such as spatial light modulator (SLM) based on electrically driven liquid crystal-based device, thermo-optical or thermo-electrical SLM, wherein the phase of the light wave transmitted through a material is modulated through the change of the temperature-sensitive refractive index of the material.
  • passive optical elements with azimuthally dependent optical properties such as a transparent window with azimuthally varying thickness, phase mask with varying optical thickness, special subwavelength gratings, metasurfaces, or deformable mirror, or active optical devices such as spatial light modulator (SLM) based on electrically driven liquid crystal-based device, thermo-optical or thermo-electrical SLM, wherein the phase of the light wave transmitted through a material is modulated through the change of the temperature-sensitive refractive index of the material
  • cylindrical vector beam generator including passive optical elements with azimuthally dependent polarization properties such as radial polarizers, half- wave or quarter- wave optical retarders, or active optical elements such as SLMs.
  • the above-stated objective of the invention is accomplished by separate treatment of the light scattered by subwavelength particles and reference light wave using vortex optical element.
  • the illumination and detection optical systems of the microscope are designed so that the reference light wave and the scattered light wave form two distinct spatial modes of light, having different mode characteristics, preferably spatial profdes.
  • the vortex optical element is positioned preferably in the Fourier plane (9 or 9a), where the cross-section of the reference light wave is by design smaller than the cross-section of the scattered light wave. This spatial separation, or partial spatial separation, in the Fourier plane enables the vortex optical element to have different effect on these two waves.
  • the size of the central region is preferably designed so that at least 20% of the reference light wave passes through the central region of the VOE, while at least 20% of the scattered light wave passes through non-uniform, azimuthally-dependent, part of the VOE.
  • the scattered light wave and reference light wave interfere and the interferometric images of the subwavelength particles, manifested as interferometric point spread functions are acquired by the detector located preferentially in the image plane of the microscope.
  • the scattered light wave transformed by the VOE into helical -wavefront beam or cylindrical vector beam in both cases have a doughnut-shaped intensity profde.
  • both types of these transformed beams have anti-symmetric optical properties (phase or polarization) with respect to the optical axis. Consequently, when the azimuthally-dependent scattered light interferes with the uniform reference light wave, the resulting vortex interferometric point spread function (iPSF) exhibits both constructive and destructive interference at certain azimuthal positions ( Figure 9).
  • the contrast, position, and shape of this vortex interferometric point spread function (viPSF) carry the information about the mass, lateral position, and axial position or orientation of the detected subwavelength particle enabling more accurate detection and tracking of these parameters.
  • the scattered light wave transformed into the helical-wavefront beam interferes with the reference light wave having planar or close to planar wavefront in the vicinity of the image plane.
  • the interferometric image of a detected subwavelength particle contains a bright spot and a dark spot coinciding with the position of constructive and destructive interference between the scattered and reference waves.
  • the occurrence of constructive or destructive interference depends on the phase difference between scatered and reference waves.
  • the azimuthal positions of the interference maximum and minimum are directly connected with the phase of scatered light wave, most importantly being dependent on axial height of the detected subwavelength particle, or, in other words, its distance from the detection surface ( Figure 9), or additional phase changes, such as phase changes induced by the interaction with the surface, plasmonic resonance phase changes in case of plasmonic scaterers, or changes related to Gouy phase.
  • quantification of the azimuthal position, or amount of rotation of the viPSF can be used to decipher the phase-related changes and separate them from the information about the size or mass of the detected particles.
  • the scatered light wave transformed into the cylindrical vector beam interferes with the reference light wave having a uniform or close to uniform polarization in the vicinity of the image plane.
  • the interferometric image of a detected subwavelength particle contains a bright spot and a dark spot coinciding with the position of constructive and destructive interference between the scatered and reference waves.
  • both beams have planar wavefronts and the occurrence of the constructive or destructive interference depends not on the phase, but on the local polarization state.
  • the azimuthal positions of the interference maximum and minimum are directly connected with the spatial distribution of polarization of scatered light wave.
  • this polarization distribution can be designed to be dependent for example on the in-plane orientation of an anisotropic subwavelength scaterer such as plasmonic nanorod ( Figure 12).
  • an anisotropic subwavelength scaterer such as plasmonic nanorod ( Figure 12).
  • the vortex optical element can be further combined with an additional optical element.
  • the additional optical element may be integrated into VOE, preferably into the central part of the VOE (in some embodiments, with uniform phase or polarization properties) ( Figure 7c, d).
  • the additional optical element may be placed in close vicinity of the VOE in the detection optical system of the microscope, typically within the distance from the vortex optical element that is not larger than 20% of the focal length of the lens preceding the vortex optical element on an optical axis of the detection optical system.
  • the additional optical elements may consist of or contain, for example, an optical fdter, such as neutral density fdter, polarizer, waveplate, (micro)-lens, or uniform or composite mirror. Examples of the additional optical elements will be provided herein below.
  • a vortex optical element for modifying properties of the output light of an interferometric scattering microscope, said output light comprising scattered light wave and reference light wave, wherein the vortex optical element is configured to transmit or reflect the output light and impart a different light property to each azimuthal position of the scattered light wave and a uniform light property to the reference light wave.
  • the property of the light waves the vortex optical element imparts is optical phase and the vortex optical element is a vortex phase mask with a topological charge equal to one or higher.
  • the property of the light waves the vortex optical element imparts is polarization and the vortex optical element is a cylindrical vector beam generator with a topological charge equal to one or higher.
  • the vortex optical element has a central region having a predetermined aperture diameter matching or exceeding the diameter of the beam of the reference light wave at the position of the vortex optical element, and the central region of the vortex optical element encodes a uniform optical property.
  • the vortex optical element has an outer region having a predetermined outer diameter matching or exceeding the diameter of the beam of the reference light wave at the position of the vortex optical element, and the outer region of the vortex optical element encodes a uniform optical property.
  • the vortex optical element can be combined with a polarizer positioned to generate a uniform polarization of the output light within a predetermined aperture diameter matching or exceeding the diameter of the beam of the reference light wave.
  • the vortex optical element can be combined with a spatial filter positioned to reduce the intensity of the reference light wave within a predetermined aperture diameter matching or exceeding the diameter of the beam of the reference light wave.
  • the vortex optical element has an area having uniform optical properties or a predetermined aperture and wherein the diameter of the area having uniform optical properties within the vortex optical element or the diameter of the predetermined aperture is larger than 1 micrometer and smaller than 10 millimeters.
  • the illuminating optical system shares at least one optical component with the detection optical system, such as a microscope objective, beamsplitter, or segmented mirror, enabling combination and separation of the illumination and detection optical paths.
  • the optical system includes a microscope objective, and the active or passive vortex optical element is integrated into the microscope objective or positioned directly behind the back aperture of the microscope objective.
  • the present invention enables direct detection of the light scattered by subwavelength particles and quantification of essential properties of the scattered light including its phase and polarization.
  • the underlying principle of the invention is the interference of the scattered light with the reference light wave in a homodyne common-path interferometer, also known as interferometric scattering microscope, or iSCAT, with a specifically designed active or passive phase, polarization, and/or amplitude filter, here denoted as vortex optical element.
  • a laser illumination source 16 is used to produce a collimated illumination light wave 5, which is focused on a back-focal plane 9 of a microscope objective 8 to illuminate the detection volume 1.
  • the position of the sample containing subwavelength particles 4 is aligned with the detection volume using sample positioning means 3, so that the detection surface 2, which is part of a glass coverslip, is located in the object plane of the microscope.
  • Subwavelength particles attach to the detection surface, resulting in their immobilization in the object plane, so that a still and focused interferometric image of these particles can be detected by the microscope.
  • the scattered light wave, and the reference light wave 7 reflected on the coverslip surface are collected with by the same microscope objective that is used for the illumination.
  • We used a beam splitter 10 to combine the illuminating optical system with the detection optical system.
  • a detection optical system comprised of the microscope objective, tube lens 11, 4-f system 12, 13 and a detector 15 is arranged so that it contains Fourier plane 9 being a back-focal plane of the microscope objective and an intermediate Fourier plane 9a within a 4-f system.
  • the distances between all neighbouring lenses along the optical axis of the detection optical system 8, 11-13 is equal to the sum the focal lengths of each pair of these neighbouring lenses.
  • a vortex optical element 14 in particular a vortex phase mask comprising a set of segments arranged around the mask and producing phase-shifts growing monotonously with the azimuthal angle from 0 to 2n radians ( Figure 8a), is introduced in the Fourier plane 9a.
  • the reference light wave 7 approaches the camera as a plane wave having a uniform phase through the beam profile, while the scattered light wave 6 is focused into a specifically shaped beam having helical wavefront and a doughnut-shaped intensity profile, thus, sometimes denoted as a doughnut beam.
  • the scattered light wave focuses towards the image plane, the overlapping fields of opposite phase shifts result in a destructive interference of the scattered light in the center of the scatered light image, while the phase of the focused scatered light wave depends on the azimuthal coordinate of the beam.
  • the reference light wave and the scatered light wave interfere on the camera giving rise to an interferogram having an intensity ID at any position of the detector corresponding to:
  • E re f and E sca t are the electric fields of the reference and scatered wave, respectively, and A ⁇ p represents the phase-shift between the waves at the position of detection.
  • a calculated interferogram of the scatered and reference fields is shown Figure 9.
  • the contrast C of the interferogram can be denoted as the difference between the intensity at the interferometric maximum and the intensity at the interferometric minimum of the interferogram, normalized to the background intensity of the reference light wave:
  • FIG. 11 depicts the measured interferogram corresponding to a single gold nanoparticle as different focal positions of the interferometric scatering microscope.
  • the change in the scatering phase results in the change in the orientation and shape of the point spread function, however the resulting maximum to minimum contrast is preserved within an extended depth of focus without any strong phase modulation.
  • the information about the relative phase can be retrieved by localizing the positions of the interferometric maximum and the interferometric minimum within the point spread function.
  • the relative phase can be deduced by estimating the angle of the vector connecting the interferometric maximum and the interferometric minimum in the image of the detected interferogram.
  • More advance data processing algorithms including an analytical or experimental fiting of the vortex interferometric point spread function or protocols based on machine learning algorithms can be implemented.
  • Figure 10 shows experimental images of silica nanoparticles of two different diameters immobilized on the detection surface resulting in an image of different scatering contrast as well as different scatering phase associated with the different axial position of nanoparticle centers of mass.
  • Figure 11 demonstrates the effect of phase detection for a single gold nanoparticle measured at different focal positions through an extended range of focus. While the phase information encoded in the orientation of the point spread function varies with the focus position of the nanoparticle the detected contrast is preserved approximately within the whole Rayleigh range of the focus.
  • the example emphasizes the versatility and robustness of the method for extracting relative phase information from interferometric data, demonstrating its applicability across diverse fields that rely on precise measurements and analytical insights.
  • FIG. 2 Alternative configurations of the interferometric scattering microscopy include embodiment with trans-illumination depicted in Figure 2 where the illumination light wave transmitted through the detection volume is used as the reference light wave in the detection optical system.
  • the detection optical system in this case can be arranged similarly to the one discussed previously ( Figure 1). Most striking difference here is that the illumination and detection optical systems are independent here and the beam-splitter can be in this case replaced with a mirror ( Figure 2).
  • Embodiments of the invention illustrated in Figure 3 and Figure 4 are based on a confocal imaging arrangement with a scanning mirror 17 ( Figure 3) or beam deflectors 19 ( Figure 4).
  • a collimated beam of illumination light 5 is focussed with the microscope objective into the detection volume.
  • the scanning mirror or beam deflectors are used to rapidly scan the position of the focused spot along the detection surface in a common scanning-focus illumination configuration, typically involving a 4-f scanning systems (details are not displayed in Figure 3 and 4).
  • the diameter of the incident beam is selected to partially fill the back aperture of the microscope objective.
  • the reflected light and scattered light are collected with the microscope objective and directed through the beam splitter, Fourier plane 9 or 9a comprising the vortex optical element and imaging optics to reach the detector, preferably a digital camera.
  • a vortex optical element featuring the uniform central aperture (Figure 7c or Figure 7d) matching the diameter of the reference beam 7 at the position of the vortex optical element or larger than the diameter of the reference beam 7 is preferred.
  • a vortex element without a central aperture Figure 7a or Figure 7b
  • the area of the vortex optical element outside of the outer diameter of vortex optical element has uniform phase or polarization properties.
  • An alternative confocal imaging embodiment shown in Figure 5 implements the beam deflector in the optical path between the beam splitter and the microscope objective.
  • a point detector 15 or a digital camera detector 15 can be used in the imaging beam path to detect the light intensity fluctuation synchronously with the focus scanning position to reconstruct the interferometric image of the specimen in the detection volume.
  • the vortex optical element is positioned in the Fourier plane 9 or 9a to modify the spatial profile of the scattered light wave.
  • FIG. 6 An alternative embodiment is shown in Figure 6, where the vortex optical element is placed close to the Fourier Plane 9, typically near the back focal plane of the microscope objective 8.
  • the vortex optical element with the uniform central region (Figure 7c, d) is preferred in the embodiment shown in Figure 6.
  • the illumination light wave 5 is focused to the back-focal plane 9 so that it is transmitted through the uniform area of the vortex optical element 14 and illuminates the detection volume.
  • the spatial profde of the scattered light wave propagating through the detection optical system is then modified by the vortex phase mask in such a way that the phase is gradually shifted in the azimuthal direction and the phase at any point of the beam is opposite to the phase at the opposite sides of the optical axis, i.e., opposite parts of the beam are phase-shifted by odd multiples of n (half a wavelength).
  • a beam has a helical wavefront and a doughnut intensity profile.
  • the image plane is further recovered along the optical path of the detection optical system to coincide with the position of the detector 15.
  • presence of the 4-f system in the detection optical system is not necessary as only one lens 13 can be used to image the subwavelength particles onto the detector.
  • the lens 13 is positioned to have one focal plane aligned with the Fourier plane 9 featuring the vortex phase mask and the second focal plane aligned with the detector 15.
  • Other conventional imaging arrangements are possible including an arrangement depicted in Figure 6, but in transmission illumination configuration analogical to one depicted in Figure 2.
  • the vortex optical element-based iSCAT microscopes illustrated in Figure 1-6 was focused on the different configurations of illuminating optical system and detecting optical system. Furthermore, in all of these configurations, the vortex optical element can be realized as an active or passive vortex phase mask or cylindrical vector beam generator and can be designed to work either in transmission or in reflection.
  • the vortex optical element can be also implemented in the form of a cylindrical vector beam generator to transform the scattered light wave into a cylindrical vector beam enabling quantitative measurement of the scattering anisotropy or orientation of subwavelength anisotropic scatterers.
  • the cylindrical vector beam generator may be realized in the form of a compound half-wave plate with monotonously changing orientation of the fast axis in the azimuthal direction. The orientation of the fast axis may be changed continuously or discretely in the azimuthal direction, see Figure 7.
  • the central region of the cylindrical vector beam generator features either a waveplate singularity or an area having uniform effect on the polarization of the light wave transmitted or reflected from it, see Figure 7.
  • a birefringent material with uniform orientation of the fast axis such as a uniform quarter-wave or halfwave retarder, or a uniform polarizer, or other optical element having spatially uniform effect on the polarization state of the transmitted or reflected light.
  • the scattered light reaches the detector in form of the cylindrical vector beam having different polarizations at different azimuthal positions of the point spread function ( Figure 8b), while the reference light wave is made uniformly polarized in the image plane, where the detector is located.
  • the reference light wave is directed through the central segment of the cylindrical vector beam generator having a diameter corresponding to or larger than the diameter of the beam of the reference light wave, typically ranging between le-3 mm and 10 mm but not larger than 75% of the diameter of the scattered light wave.
  • a slight off-axis positioning of the cylindrical vector beam generator ensures that the reference beam avoids the central singularity of the cylindrical vector beam generator and passes through an area with spatially uniform polarization properties.
  • the interference of the cylindrically polarized scattered light wave and uniformly polarized reference light wave, preferably a linearly polarized reference light wave, in the image plane on the detector results in a vortex interferometric point spread function having a distinct bright maximum and distinct dark minimum in the positions of constructive and destructive interference.
  • the polarization of the scattered light can thus be quantified by fitting the maximum and minimum of the vortex interferometric point spread function and calculating the angle between these two extremes, Figure 12.
  • the collimated scattered wave 6 having larger diameter at the Fourier plane 9a was reflected from a highly reflective aluminium surface being present on the whole mirror except of its central part. Both the scattered light wave and reflected light wave were reflected by this compound mirror and transmitted through the lens 13 to interfere in the image plane of the microscope coinciding with the position of the detector 15, where the resulting interference of cylindrically polarized scattered light wave and linearly polarized reference light wave was detected.
  • the point spread function orientation in Figure 12 highlights the polarization of the scattered wave determined by the rotational orientation of the subwavelength scatterers.
  • the vortex optical element can be further combined with a spatial filter optimized to reduce the intensity of the reference light wave E re f resulting in an increase of the detected contrast C.
  • a partly reflective coating is applied to the central region of the vortex optical element within a predefined aperture diameter.
  • This predefined aperture diameter corresponds to or is larger than the diameter of the beam of the reference light wave.
  • the vortex optical element featuring the uniform central aperture ( Figure 7c or Figure 7d) can be designed to host the reflective coating acting as the spatial filter by depositing the partially reflective coating within the area of the uniform central aperture.
  • the present invention provides an innovative solution for interferometric scattering microscopy and mass photometry applications.
  • the integration of a phase mask into the imaging part of the interferometric scattering microscope creates a vortex interferometric point spread function, which encodes phase information and enables differentiation of the amplitude and the phase of scattered light from detected particles, such as single molecules.
  • the integration of the special half-waveplate encodes the polarization information of the scattered wave into a quantitative image of the point spread function orientation.
  • the invention has the potential to greatly advance the study of single cells, cell aggregates, lipid nanoparticles, liposomes, viruses, exosomes, and tissues, as well as contribute to a more profound understanding of biological phenomena.
  • the present invention represents a significant advancement in the state of the art.

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Abstract

La présente invention concerne un appareil de microscope à diffusion interférométrique approprié pour la détection optique quantitative de la taille, de la position 3D et/ou de l'orientation de particules de sous-longueur d'onde individuelles dans un échantillon, ledit microscope comprenant un élément optique à vortex configuré pour conférer différentes valeurs d'une propriété de lumière, à différentes positions azimutales de l'onde de lumière diffusée et pour conférer une valeur uniforme de la propriété de lumière à l'onde de lumière de référence. En outre, l'invention concerne un procédé de détection de particules de sous-longueur d'onde à l'aide d'une microscopie à diffusion interférométrique utilisant ce microscope.
PCT/CZ2025/050009 2024-01-24 2025-01-24 Procédé et appareil de détection optique quantitative de la taille, de la position 3d et/ou de l'orientation de diffuseurs de sous-longueur d'onde individuels Pending WO2025157332A1 (fr)

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