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WO2022117695A1 - Système et procédé d'imagerie optique à super-résolution - Google Patents

Système et procédé d'imagerie optique à super-résolution Download PDF

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Publication number
WO2022117695A1
WO2022117695A1 PCT/EP2021/083882 EP2021083882W WO2022117695A1 WO 2022117695 A1 WO2022117695 A1 WO 2022117695A1 EP 2021083882 W EP2021083882 W EP 2021083882W WO 2022117695 A1 WO2022117695 A1 WO 2022117695A1
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WIPO (PCT)
Prior art keywords
sample
particles
resolution
images
light
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Inventor
Lucas Pelkmans
Ola SABET
Yanic HEER
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Zurich Universitaet Institut fuer Medizinische Virologie
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Zurich Universitaet Institut fuer Medizinische Virologie
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Publication of WO2022117695A1 publication Critical patent/WO2022117695A1/fr
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    • 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
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • G01N21/6458—Fluorescence microscopy
    • 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
    • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/0052—Optical details of the image generation
    • G02B21/0076—Optical details of the image generation arrangements using fluorescence or luminescence
    • 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
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6408—Fluorescence; Phosphorescence with measurement of decay time, time resolved fluorescence
    • 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
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/648—Specially adapted constructive features of fluorimeters using evanescent coupling or surface plasmon coupling for the excitation of fluorescence

Definitions

  • the invention relates to an imaging system for microscopic far field superresolution imaging, as well as to a method for superresolution.
  • Superresolution microscopy is known in the art.
  • Superresolution microscopy refers to a method of microscopy that allows for generating an image of a sample comprising luminescent emitters that has a spatial resolution higher than the optical resolution of the optical system it has been acquired with.
  • the term superresolution microscopy refers to far field superresolution microscopy in distinction to near-field microscopy.
  • the key for achieving superresolution is to encode information on the emitter distribution in the sample by means of a switchable optical property that the emitters exhibit.
  • the encoding is facilitated by an emitter property that allows the emitters to switch between a bright (on) and a dark (off) state.
  • the on- and off- states have to be controlled in terms of their duration and switching rate.
  • the optical volume is defined as the volume that corresponds to the optical resolution of the imaging system. Often, this volume is a diffraction-limited volume. In some cases, however, diffraction-limited imaging is not achieved.
  • each emitter in each image of the series that provided a signal will be represented by a spatially isolated point spread function (PSF).
  • PSF point spread function
  • the position of emitters of the subset of emitters that is in its on state is determined by determining a center of the PSF in terms of its highest intensity. The center can be determined with higher accuracy than the width of the PSF.
  • the coordinates of all successful localizations of PSFs might then be combined to arrive at the high-resolution image, which resolution is particularly limited by the number of photons and which resolution is higher than the optical resolution of the imaging system.
  • SMLM single-molecule localization microscopy
  • control of the switching rate and duration of the emitters is achieved by either selecting very specific fluorophores or by adding chemical compounds to the sample that are configured to adjust the switching behavior.
  • An object of the present invention is to provide a method and an imaging system that does not, or only minimally, compromises time for space, that has a low propensity for photo-bleaching, and that can scale between the nanometer and millimeter range covering tens of thousands of cells.
  • an imaging system for microscopic far-field superresolution imaging having an optical resolution, wherein the imaging system comprises at least the following components: - An excitation light source configured to provide excitation light, particularly coherent excitation light within a predefined excitation wavelength range,
  • a first objective arranged and configured to illuminate a sample comprising emitters that are excitable with the excitation light and that emit emission light upon excitation with excitation light, wherein the sample is arranged or may be arranged in a sample space of the system,
  • sample carrier arranged and configured to support the sample in the sample space, wherein the sample carrier comprises a first transparent window for supporting the sample, wherein the sample carrier is arranged such that excitation light from the first objective first propagates through the first transparent window of the sample carrier before reaching the sample space,
  • a far-field detection optics configured and arranged such in the system that emission light is collected from the sample space and propagated towards an image space along a detection path
  • a filter element arranged in the detection path and configured to filter the collected emission light from the far-field detection optics
  • a detector arranged in the image space and configured to record the emission light, particularly the filtered emission light from the sample,
  • a colloidal suspension particularly a transparent colloidal suspension, comprising particles as the colloid, particularly wherein the particles are dielectric particles, wherein the particles exhibit light scattering, absorbing, and plasmonic properties in the excitation wavelength range, wherein the suspension is arranged in the sample space or between the first window of the sample carrier and the first objective, such that excitation, particularly an excitation strength or an excitation cross-section, of the emitters with excitation light varies randomly both spatially and temporally at the positions of the emitters, due to the light scattering, absorbing and/or plasmonic properties of the particles undergoing Brownian motion in the suspension, such that light emitted from the emitters also varies randomly both spatially and temporally due to the random excitation.
  • the imaging system allows for producing and controlling temporally and spatially variant emitter signals, without the need of addition of chemical compounds to the sample in order to control a fluctuating emission behaviour of the emitters.
  • the fluctuations (magnitude and frequency) in the emission light are for example adjustable by means of a concentration adjustment of the particles in the suspension.
  • the system offers a large degree of freedom in terms of where the suspension of particles can be placed with respect to the sample.
  • sample may not to be understood to form a mandatory part of the system, but is solely used in the context of the current description and claims to illustrate the arrangement of components or the architecture of the system and/or the working principle of the method.
  • the temporally and spatially variant excitation and emission is particularly not induced by emitter quenching processes.
  • the particles are particularly nanoparticles.
  • the particles are colloidal particles.
  • the particles comprise or are silver (Ag) particles, particularly colloidal silver particles.
  • the emitters might comprise fluorophores, such as organic fluorophores or genetically encoded fluorophores.
  • the emitters are particularly luminescent emitters that are excitable by the excitation light and emit on a different wavelength than the excitation light, such that the emission light can be filtered by a wavelength dependent filter.
  • the detector is configured to sample the detected light at a timescale that is in the range or faster than the diffusion time of the particles required to diffuse through a volume of the size of the optical resolution of the system, such that subsequent recorded signals by the detector comprise an information at least on the temporal and/or the spatial correlation of the random excitation caused by the Brownian motion of the particles.
  • the array detector is configured to acquire images such that variations, i.e. fluctuations of the emitted light in the pixels of the detector are correlated in space and time, as the frame rate may be adjusted such that the diffusion rate of the particles through a volume corresponding to a pixel in the sample space is smaller than the frame rate.
  • variations i.e. fluctuations of the emitted light in the pixels of the detector are correlated in space and time
  • the frame rate may be adjusted such that the diffusion rate of the particles through a volume corresponding to a pixel in the sample space is smaller than the frame rate.
  • a concentration of the emitters is chosen such that an emitter density conforms to a sparsity criterion for compressed sensing methods.
  • the imaging system comprises a microscope that is configured to illuminate the sample by means of a wide-field illumination with the excitation light.
  • the term wide-field illumination thereby comprises illumination of the sample by conventional wide-field illumination, small angle illumination, and/or total internal reflection (TIR) illumination.
  • the system is further configured to image the sample in the sample space in a parallel fashion on the array detector, that is sensitive enough to detect photons from single emitters.
  • array detectors are for example CMOS cameras, or electron multiplying CCD cameras (EMCCD cameras).
  • the imaging process involves the signal from the emitters to be optically convolved with the PSF of the system that eventually defines the optical resolution of the system.
  • the excitation light source might be a coherent light source, such as a laser or lasingbased light source.
  • the suspension of dielectric particles with plasmonic properties introduces stronger fluctuations in the excitation light than other particles lacking the plasmonic property without compromising overall intensities.
  • the system particularly comprises a beamsplitter that is arranged such in the light path of the system that excitation light can be filtered from the emitted light.
  • the system can further comprise an emission filter that blocks any excitation light to a higher degree than the beamsplitter.
  • the excitation light and the emission light of the emitters at least partially comprise a different wavelength.
  • the array detector might comprise an optical assembly, e.g. an imaging lens, that allows for magnified imaging the sample on the array detector.
  • an optical assembly e.g. an imaging lens
  • the system might comprise further optical elements configured to shape, filter or alter the wave front of the excitation and/or emission light.
  • the system is particularly arranged and adapted to illuminate the sample simultaneously and not by means of a point- or line-scanning method.
  • the system is arranged and adapted for confocal excitation and/or confocal detection, such as a spinning disk wide field detection microscope or a confocal point detection microscope, particularly wherein the detector is a point-detector.
  • confocal excitation and/or confocal detection such as a spinning disk wide field detection microscope or a confocal point detection microscope, particularly wherein the detector is a point-detector.
  • the microscope is a confocal laser scanning microscope.
  • the sample carrier may comprise a sample holder that may have a sample chamber for immersing the sample with the suspension of particles.
  • the first transparent window is a coverslide or a cover slip having a thickness of only around hundred to two hundred microns.
  • the detection optics might be comprised in the first objective, such that illumination and collection of light is achieved by the same objective.
  • the detection optics it might be provided by means of a second objective that is arranged and configured to collect the emitted light at a different angle than the first objective.
  • Such imaging system therefore may include a light sheet imaging modality.
  • Using a second objective for detection particularly allows that the emitted light from the emitters does not propagate through the suspension on its way to the detector.
  • the suspension of particles comprises a transparent liquid surrounding the particles
  • the suspension is transparent at least within a wavelength range between 300 and 800 nm.
  • transparent particularly comprises the notion of a hued, colored or tinted suspension, that is the suspension exhibits wavelength-dependent absorption properties.
  • the dielectric particles have a size such that they exhibit plasmonic resonance with the excitation light. For example, particles of 50 nm in diameter will show strong interactions with excitation light at a wavelength of 488 nm.
  • the particles are not bound or are connected to the emitters or a vicinity of the emitters.
  • the particles are inert particles in terms of a chemical reaction taking place with the emitters.
  • the optical resolution particularly the optical resolution of the acquired images of the imaging system is diffraction limited.
  • the Brownian motion of the particles causes spatial correlations, particularly on a length scale smaller than the diffraction limit and/or the optical resolution of the system, in the excitation of the emitters, allowing a varying excitation on length scales that are shorter than the optical resolution of the imaging system such that there is stronger incoherence in the excitation of the emitters.
  • the interaction of the excitation light and the particles cause the generation of a spatio- temporally varying speckle illumination of the sample space, particularly wherein a speckle size of the speckles is diffraction limited.
  • the system allows for generation of image sequences that in which the speckles spatio-temporally vary in the images of the sequence, such that a compressed sensing method may be reconstruct a super-resolved image despite the speckle size being in the diffraction range and thus not in the sub-diffraction range.
  • a concentration of the particles in the suspension is adjusted such that in a volume corresponding to the optical resolution of the imaging system in the sample space not more than 10 particles, particularly not more than 5 particles, more particularly not more than 1 particle are present at the same time on average.
  • This embodiment allows for the system to acquire images of the sample that exhibit intensity fluctuations that are neither averaged out during the acquisition of a single image (or supress the signal of the emitters completely) due to a too high concentration of the particles, nor do the signals only fluctuate seldomly in a series of images which would prolong acquisition times.
  • the concentration of particles is particularly adjusted such that on average more than one, particularly more than 5 emitters in a volume corresponding to the optical resolution, e.g. a diffraction limited volume, are excitable by the excitation light or the plasmonic particles.
  • This embodiment stands in contrast to single-molecule localization methods that rely on a lower average number of emitters signalling, i.e. being in an on-state, in a diffraction limited volume.
  • the array detector has an adjustable frame rate with a corresponding reciprocal frame time, at which the array detector may record the images from the sample, wherein a viscosity of the suspension is adjusted such that a diffusion time of the particles in the suspension through a volume corresponding to the optical resolution of the imaging system in the sample space is in the same order of magnitude or longer than the frame time, particularly wherein the diffusion time is not shorter than a tenth of the frame time, particularly not shorter than a fifth of the frame time.
  • volume corresponding to the optical resolution of the imaging system has to be understood, as a volume that is defined by the optical resolution of the imaging system. The optical resolution might differ along the three dimensions of space in the sample, and even might be locally varying due to varying sample properties or limitations inherent to optical components of the imaging system.
  • said volume corresponds to the diffraction limited volume.
  • This embodiment allows a well-adjusted camera frame rate with regard to the temporal fluctuations in the sample.
  • the frame rate of the camera is too slow, the fluctuating signals are averaged within a single frame, i.e. image, and thus the information content about the positions of the emitters is reduced, leading to longer acquisition times.
  • the camera frame rate is too fast with regard to the fluctuation time scale and thus the diffusion rate, the intensity of the signal of the emission light is reduced in each image, which causes a higher amount of noise being present in each image, which too, reduces the information content with regard to the emitters positions.
  • the particles are smaller than 130 nm in diameter, particularly smaller than 100 nm, more particularly smaller than 75 nm, even more particularly larger than 40 nm or 30 nm in diameter.
  • the particles have an average size in the range of 30 nm to 100 nm.
  • the particles have an average diameter between 50 nm to 75 nm.
  • average diameter particularly refers to the average of diameters of a plurality of particles.
  • diameter particularly refers to an average hydrodynamic diameter in the suspension or to an average diameter of the particle. It is noted that the particles do not have to be round, in order to appropriately define the diameter. For example, the diameter of the particle is the diameter that would correspond to a sphere having the same volume as the particle. If the particles are smaller than 20 nm their optical properties regarding absorption, scattering or plasmonic excitation might become too low in order to induce the fluctuation in the emitter emissions.
  • the particles comprise a dielectric compound or wherein the particles comprise or consist of a metal compound or a metal alloy, such silver and/or gold, particularly wherein the particles are silver particles or wherein the particles are gold particles.
  • silver is known to exhibit plasmonic excitation properties in the visible light range, particularly such that excitation is altered depending on the distance of the particle to the emitter.
  • Gold particles in turn might be suitable for two-photon excitation and/or for imaging in the red to infrared spectral region.
  • the suspension has a viscosity that is at least 20 times higher than the viscosity of water under the same conditions, e.g. at room temperature and normal atmospheric pressure, and/or wherein the suspension has a refractive index, wherein said refractive index is within a range of ⁇ 20% of the refractive index of the window, particularly within a range of ⁇ 20% of the refractive index of glass, particularly within 1.2 and 1.8.
  • the diffusion rate of the particles can be adjusted such that frame rate of the array detector and diffusion rate of the particles through a volume corresponding to the optical resolution allow a best possible resolution of the fluctuations in the recorded signals.
  • the refractive index of the suspension matches the refractive index of the window, such that optical aberrations causing a deterioration of the optical resolution of the system caused by a refractive index mismatch between the window and the suspension are kept low.
  • the first objective is an immersion objective, having an immersion medium arranged between the objective and the sample carrier, wherein the immersion medium consists of the suspension with the dielectric particles.
  • This embodiment allows for imaging a sample at high-resolution, without the need to apply the suspension to the sample.
  • a liquid immersion medium for oil or water immersion objectives for optical microscopes wherein the immersion medium is, or comprises the suspension with the particles, particularly wherein said suspension has the refractive index of water or the refractive index of immersion oil, particularly the refractive index of glass, more particularly a refractive index in the range of 1.50 and 1.53, particularly at room temperature.
  • the concentration, composition and optical properties of the particles are disclosed for the embodiment relating to the system and are applicable to the immersion medium as well.
  • the particles exhibit light scattering, absorbing, and/or plasmonic properties upon exposure to excitation light, such that spatio-temporal fluctuations in an excitation light passing through the liquid immersion medium may be generated.
  • the sample carrier comprises a second transparent window arranged opposite the first transparent window, wherein the second window faces, i.e. is closer to the first objective and the first window faces, i.e. is closer to the sample space, wherein the suspension is arranged in a space between the first and the second window.
  • the sample carrier allows for superresolution imaging of the sample, without having the suspension in direct contact to the sample or the first objective.
  • the first objective is an oil immersion objective, used with an immersion oil
  • no additional refractive index mismatches are induced by the addition of the second window.
  • the refractive index of the suspension is adjusted as well to the refractive index of the immersion oil, no additional aberrations are induced at all, by the sample carrier.
  • the first and the second window are essentially stacked on top of each other along the optical axis, while preserving a gap that comprises the suspension.
  • the gap has an equal height throughout the first and the second window, i.e. the windows extend parallel to each other.
  • the first and the second transparent window are comprised by a container, wherein a container top and bottom side comprise the first and the second window respectively, wherein the container comprises a container wall at its lateral portions that seals the suspension in the space between the first and the second window.
  • the container is comprised in the sample carrier.
  • the gap is in the range of one to 200 microns, particularly wherein a thickness of the first and the second window are within 50 to 200 microns.
  • a container for superresolution imaging comprising a top and bottom side which comprise the first and the second window respectively, wherein the container comprises a container wall at its lateral portions that seals the suspension of particles in the space between the first and the second window.
  • the second window faces, i.e. is closer to, the first objective and the first window faces, i.e. is closer to, the sample space.
  • the space has a height in the range of one to 200 microns, particularly wherein a thickness of the first and the second transparent window are within 50 to 200 microns.
  • the height of the container particularly extends along the optical axis, when the container is arranged in the imaging system.
  • the container comprises a second transparent window arranged opposite the first window, wherein the second window faces, i.e. is closer to the first objective and the first window faces, i.e. is closer to the sample space, wherein the suspension is arranged in the space between the first and the second window.
  • the properties of the suspension such as viscosity and medium, and the particles, such as material, optical properties and sizes, can be adjusted or chosen as described throughout to specification even if related to other embodiments and aspects of the invention.
  • the liquid of the suspension comprises or consists of 97% 2,2’-thiodiethanol (TDE).
  • the container is comprised by a or the sample carrier.
  • the container is comprised by a microscope objective.
  • the detection optics comprise a second objective configured and arranged to collect the emitted light from the sample in the sample space and to propagate the emitted light from the sample to via the detection path and the filter element to the array detector.
  • This embodiment allows for example for light sheet imaging, particularly in a 90° configuration.
  • the excitation path and the emission path of the imaging system are completely separate light paths.
  • the system comprises a computer connected to the detector, wherein the computer is configured to receive data from the detector, wherein the data comprise information about a series of recorded images of the detector, wherein in at least some pixels of the images a fluctuating signal, particularly a fluctuating sum signal, is comprised that originates from fluctuating emissions and/or fluctuating excitations of a plurality of emitters that are imaged to the at least some pixels of the images, particularly wherein said fluctuating emissions and/or said fluctuating excitations are caused by the random moving particles in the suspension, wherein the computer is configured to process the data by means of a compressed sensing method that is configured to output a high-resolution image of the sample that has a spatial resolution better than the optical resolution of the system.
  • a fluctuating signal particularly a fluctuating sum signal
  • This embodiment allows for generating a superresolution image by means of a compressed sensing method, with the system according to the invention.
  • the compressed sensing method is configured to evaluate the recorded fluctuations of the series of images, and to determine the high-resolution image.
  • the series of images comprises not more than 1.000 frames, particularly not more than 100 frames, more particularly not more than 50 frames, wherein from that series the determined high-resolution image is generated to completion, meaning that the high-resolution image is not generated only partially.
  • the determined high- resolution image for example comprises information on emitter positions not only of a fraction of the emitters but for all emitters that contributed to the evaluated signal sufficiently and/or the Nyquist criterion for sampling a structure of the emitters in the sample is met.
  • the imaging system comprises a motion-inducing device that is configured to prevent sedimentation of the particles in the suspension, particularly wherein said motion-inducing device comprises a shaker, an ultrasonic sound source and/or an electric and/or a magnetic field generating device, wherein said electric and/or magnetic field is configured to induce motion of the particles.
  • the motion-inducing device is connected to or arranged at the container or at the sample carrier.
  • a method particularly a computer- implemented method, a computer program comprising computer program code or a computer program product, for superresolution imaging with a microscope imaging system, particularly with an imaging system according the first aspect of the invention, is disclosed, said method, computer program or computer program product being configured to image a sample or to acquire data of the imaging of the sample having emitters that are excitable by excitation light and that are located at the sample, particularly fixedly attached to the sample, wherein the method comprises the steps of:
  • a colloidal suspension particularly a transparent colloidal suspension, comprising particles as the colloid in the sample space or between the first window of the sample carrier and the first objective, wherein the particles exhibit light scattering, absorbing, and/or plasmonic properties in the excitation wavelength range, such that excitation, particularly an excitation strength or an excitation cross-section, of the emitters with excitation light varies randomly both spatially and temporally at the positions of the emitters, due to the light scattering, absorbing and/or the plasmonic properties of the particles undergoing Brownian motion in the suspension, such that light emitted from the emitters also varies randomly both spatially and temporally due to the random excitation.
  • the computer program may be stored as a compute product on a non-volatile storage medium, that when executed by a computer executes the method according to the invention, particularly the step comprising the compressed sensing method. Particularly, after executing or during execution of the computer program at least one of the series of high-resolution images is displayed or an average of the high-resolution images.
  • the particles are dielectric particles.
  • the fluctuations that are induced in the series of images by the randomly moving particles in the suspension are expected to be Gaussian or Poisson distributed with respect to their temporal behaviour around an average or mean fluctuation time.
  • the term fluctuations in the images particularly relates to temporal changes in intensity in the pixels of the image over time.
  • the pixel As the pixel’s intensity changes in the plurality of pixels, the fluctuations are spatio-temporal fluctuations.
  • compressed sensing method refers to a computer-implemented method that is configured to reconstruct a high-resolution image from the recorded images, wherein the recorded images, particularly the sample structure and the emitter density in the sample, suffice the sparsity requirement necessary for successfully compressed sensing.
  • a computer For executing the compressed sensing method, a computer with a processor and a storage device for storing the series of images is provided.
  • the computer comprises a processor on which the compressed sensing method may be executed.
  • the images might be transmitted from the array detector to the computer in form of digital data.
  • the particles of the suspension comprise particles made of a dielectric compound or wherein the particles comprise or consist of a metal compound or a metal alloy, such as silver and/or gold, particularly wherein the particles are silver or gold particles.
  • Particularly silver is known to exhibit plasmonic excitation properties in the visible light range, such that excitation is altered depending on the distance of the particle to the emitter.
  • Gold particles in turn might be suitable for two-photon excitation and/or for imaging in the red to infrared spectral region.
  • an average low-resolution image y m is determined from an average or a sum of the images yr of the acquired series, and wherein the compressed sensing method determines an average high-resolution image x m corresponding to an average or a sum of the series of high-resolution images Xf.
  • the average low-resolution image may be determined from a temporal mean value for each pixel of the images of the acquired series.
  • the average high-resolution image may be determined from temporal mean value for each pixel of the images of the series of high-resolution images.
  • the average high-resolution image is well-suited to relay the generated information of the increased spatial resolution of the sample to a user, for example by displaying the average high-resolution image, particularly together with the low- resolution image.
  • each image of the acquired series has a width of / pixels and a height of j pixels, particularly wherein the pixels are arranged in pixel rows and pixel columns of the image, wherein each pixel has a pixel value and comprises information about an area in the sample from which light is imaged onto the pixel, wherein a sampling factor s is selected that is larger than 1 , and is for example an integer number, wherein each image of the series of the determined t high-resolution images Xf, has /*s pixels along a width of the high-resolution images and j*s pixels along a height of the high-resolution images.
  • the high-resolution images and particularly the average high-resolution image are determined by the compressed sensing method by means of a convex optimization method, wherein the deviations between the series of the determined high-resolution images Xf and the acquired series of images yr are determined by applying the Euclidian norm
  • A is a matrix, that might comprise information on the point spread function of the imaging system
  • y corresponds to a vector comprising the pixel values of the images yr of the acquired series of images and particularly the average low-resolution image y m
  • x corresponds to a vector comprising the pixel values of the high- resolution images x f and particularly the average high-resolution image x m
  • the convex optimization method determines the matrix A and x such that
  • s is a positive threshold value, particularly wherein s is determined from a noise comprised in the images, such that s indicates a limit to the convex optimization method.
  • the matrix A (also referred to as sensing matrix in the context of the current specification) comprises and reflects a localized spatio-temporal randomness arising from the Brownian motion of particles in the suspension and their effect on light over the varying speckle pattern illuminations that cause the fluctuations in the signal.
  • the sparsity prior for the compressed sensing method enhances an achievable spatial/optical resolution beyond the two-fold enhancement of structured illumination microscopy (SIM), and the number of measurements, i.e. the number of recorded low resolution images, required to recover the unknown x is drastically reduced due to the efficient randomness of the sensing matrix A.
  • SIM structured illumination microscopy
  • the compressive sensing method satisfies the randomness requirement. This allows to overcome the well-known tradeoff between achievable resolution and measurement time.
  • the threshold value s is indicative of a noise characteristic of the recorded images, wherein the noise characteristic is determined from noise of the array detector and a Poisson noise of the detected light on the array detector.
  • the determination reflects a physical meaningful limit for resolution increase, such that an over-restoration of the high-resolution images is prevented.
  • the average high-resolution is displayed on a display.
  • the point-spread function of the imaging system is acquired in a separate measurement.
  • the PSF might be determined with a very high signal-to-noise ratio from a different measurement with a sample dedicated to the PSF measurement.
  • a sample might comprise separately arranged bright emitters, such as fluorescent beads comprising a plurality of single fluorophores.
  • the PSF is determined as a function of location of a field of view of the system, particularly as a function of location in the images, such that a locally varying PSF can be used for determining the high-resolution images. This in turn leads to even better restoration results.
  • the problem is solved by a computer program, comprising computer program code that when executed executes the compressed sensing method according to the invention.
  • the invention provides superresolution imaging by exploiting the mathematical theory of compressed sensing (CS) that builds on the fact that most images can be described by a number of parameters much lower than the total number of pixels.
  • CS leverages on this low mathematical complexity (sparsity) in order to recover images from far fewer measurements than normally required.
  • the method according to the invention allows for computationally generating superresolved images from a sequence of diffraction-limited images.
  • each pixel of the image measures the compressed form of a spatially random subset of sub- diffraction-modulated true signal.
  • the signal measured by each low-resolution pixel can be spatially decompressed into multiple pixels, effectively creating a new image with a higher sampling rate and an increased spatial resolution.
  • the term low- resolution (LR) pixel refers to the pixels of the images acquired by the array detector, i.e. the images that are provided to the method in order to increase the resolution.
  • the term low-resolution image is also referred to as the acquired image in the context of the current specification, i.e. the LR frame or image is an image acquired with the imaging system and has a spatial resolution that corresponds to the optical resolution of the imaging system.
  • y Ax.+noise
  • y the vector form of the series of low-resolution images
  • A the sensing matrix describing the linear mapping from the source to the observation, containing information about the illumination modulation that may comprise the spatio-temporal varying speckle patterns generated by the suspension and the PSF
  • x the vector from of the high-resolution images that are to be determined
  • noise represents the measurement noise.
  • x can be resolved by putting a set of constraints on the sensing matrix and x, while minimizing the real non-negative function, minimize
  • each SR image in x comprises s x s more pixels than each low-resolution image y.
  • a virtual grid of s * s pixels on each pixel of each low-resolution (LR) image is overlaid or generated.
  • the pixels of the virtual grid of s x s pixels are referred to as superresolution (SR) pixels.
  • SR superresolution
  • Each super resolution pixel models the contribution of the luminescence, particularly the fluorescence coming from the emitters correspondingly located in the sample space within that pixel. Therefore, calculating the correct intensities of every pixel in all SR-images represents a higher resolution reconstruction (Fig. 9).
  • the method according to the invention computes the SR pixel intensities for all frames at once, by solving a comparably large convex optimization problem, for which a modified version of the solver SCS [10] driven by a MATLAB interface code YALMIP [11] can be used.
  • the minimization problem is converted to a convex program with a linear objective function, and the solution may be found using an established first-order solver [2] (Fig. 1).
  • a solution /W-component vector x encoding the reconstructed SR image is prepared; x is the concatenation of three smaller vectors x M , x F , x B that are defined as follows:
  • ⁇ x M x M (/, j) are the w*s*/?*s unknowns representing the SR solution image.
  • ⁇ x F x F (/, j, t) are w*s*h*s*T unknowns capturing the effect of particle (particularly silverj-induced intensity modulation: they represent the difference between a reconstructed pixel in frame t and its corresponding time average.
  • These are auxiliary variables that are used in formulating the convex optimization problem but do not otherwise contribute to the final SR output.
  • ⁇ x B (x B (1), x B (2), x B (3), x B (4)) is an optional 4-vector encoding non-specific background illumination: four illumination sources are distributed (evenly spaced) across the border of the reconstruction region. For every measured pixel the predicted background contribution is a distance-weighted average of the four intensities x B (1), x B (2), x B (3), x B (4).
  • the optimization problem is to minimize the objective function, subject to the following constraints:
  • ⁇ x and y are previously defined.
  • ⁇ s is the reduced chi-square target defined in the section "Noise model" and corresponds to a noise characteristic of the array detector.
  • ⁇ A P*Q*R is a matrix (N * M) formed by the matrix product of:
  • R which is an M x M matrix used to vary the weight of particle-mimicking variables x F in the problem by randomly multiplying each variable with either -1 (means the pixel emits less photons than its value in x M ) or +1 (means the pixel emits more photons than its value in x M ).
  • -1 meanans the pixel emits less photons than its value in x M
  • +1 meanans the pixel emits more photons than its value in x M .
  • the net effect is that the particle-mimicking variables can have a negative contribution due to this randomization of signs.
  • R acts as the identity matrix.
  • the solver SCS may be modified to handle convolution with the PSF using FFT (Fast Fourier Transform) instead of matrix-vector products.
  • FFT Fast Fourier Transform
  • Constraint 4 Lower bound values for Aq-mimickinq variables (Constraint 4): by the same reasoning as for Constraint 3, a constraint may be imposed such that x F (/, j, f) > 0.05 * x M (/, /) ⁇
  • Density constraint (Constraint 5) stems from the motivation to impose a structure on the solution. The idea is that there should be no pixel pairs (H, j1) and (i2, j2) which are resolved in the solution if their distance is less than the theoretically achievable resolution supported by the method according to the invention.
  • a synthetic template reference library (pixel size 18 nm) composed of 11 pairs of input signals, i.e. emitters, is provided, wherein the pairs of emitters are separated by distances ranging from 36 to 18 nm (Fig. 1 B).
  • 12 runs are simulated, wherein each run comprises a series of 100 diffraction-limited images, i.e. low-resolution images at pixel size 72 nm (the typical pixel size in an sCMOS camera at 90x magnification) using either an intensity modulation scheme, or no modulation as control. Camera noise and Poisson photon noise are added to the sequence of images.
  • the intensities of the emitters in the template are varied randomly by ⁇ 30% relative to a mean intensity of the emitters.
  • Reconstruction using pixel size 18 nm resolves separations down to 36 nm, which is the smallest theoretically separable distance in the library, but only when intensity modulation was applied (Fig. 2, left graph).
  • the method according to the invention is executed on computationally simulated microtubules (Fig. 4). After generation of 20 low-resolution images at pixel size 72 nm with intensity modulation, said images are reconstructed into super-resolved (SR) images using grid pixel sizes of 36 or 24 nm. Line profiles through selected filaments showed that the method according to the invention resolves closely aligned microtubules, and that filaments that cannot be well separated with pixel size 36 nm can be resolved better with pixel size 24 nm. Additionally, Pearson correlation analysis shows that reconstructions errors in dense regions decrease as the sampling frequency increases, i.e. smaller pixel size/ higher scale factor for the SR images. Thus, by increasing the sampling factor s used during decompression, the spatial resolution in the SR image can be increased.
  • the corresponding imaging system comprises far-field optics microscope, such as a TIRF or Widefield fluorescence microscope with an array detector, for example in form of a light sensitive camera that is configured to detect fluctuating light from single emitters.
  • far-field optics microscope such as a TIRF or Widefield fluorescence microscope with an array detector, for example in form of a light sensitive camera that is configured to detect fluctuating light from single emitters.
  • array detector for example in form of a light sensitive camera that is configured to detect fluctuating light from single emitters.
  • Ag particles 502 of such dimensions exhibits strong, wavelength-dependent scattering and absorption of light that may be exploited by placing said randomly moving particles in the light path close the sample (Fig. 5).
  • the light intensity reaching the emitters 506 at the sample is lowered due to the particle 502 and the corresponding fluorescence emission of these emitters is lowered too, while neighboring emitters that remain exposed to the full amount of light will not be affected.
  • the second CS requirement, randomness in signal sampling is achieved by the high viscosity of 97% TDE, which slows down Ag particle motion, ensuring that movement by Brownian motion primarily occurs between consecutive frames.
  • the concentration of the Ag particles is adjusted such that enough random illumination events are obtained for standard fluorophores without strongly affecting the overall signal-to-noise ratio of the signal. Acquiring a sequence of LR images with this setup subjects the signal that each pixel samples to a unique spatiotemporal light modulation (dynamic modulation) scheme.
  • Using as few as 20 frames of the acquired LR images allow reconstruction the low- resolution information into a superresolution image, choosing a sampling factor of 2, i.e. a pixel size of 36 nm.
  • Line profiles through 25 microtubule filaments show an average width of ⁇ 80 nm when using Ag, compared to -143 nm when not using Ag, demonstrating that the method enables superresolution microscopy even with a small sampling factor of 2 and without the need for a prohibitively large amount of Ag particles (Fig. 7). Since Ag particles of diameter 55-75 nm have the potential to modulate light of multiple wavelengths, the method may be applied to multi-color experiment using emitters that emit in different wavelength ranges.
  • COS-7 cells are transfected with an outer mitochondrial membrane marker mEGFP-TOM20, and mitochondrial dynamics are imaged at high speed and at superresolution for extended periods of time (Fig.8).
  • 1,000 consecutive images were taken at a frame rate of 25 frames/s, and two modes were used for super-resolved image reconstruction.
  • the first mode 20 consecutive frames are used for reconstruction into one super-resolved average image of 36 nm pixel size, resulting in a time interval of 800 ms in the final time-lapse sequence.
  • super-resolved images are generated using a sliding window of 10 frames every 5 frames, resulting in a time interval of 200 ms in the final time-lapse sequence.
  • the high spatial resolution of the method according to the invention ensures detection of densely packed mitochondria. Additionally, it is possible to capture mitochondrial dynamics at very high temporal resolution across a large field of view (Fig. 8), providing high spatial detail of fast-moving mitochondria-derived vesicles. Even though some photobleaching might be observed over the 100 frames, the reconstruction was robust to changes in overall intensities, indicating a proper modeling of the measurement noise in each pixel as the sum of Poisson photon count noise and camera readout noise. Such processes cannot be captured with a 3- fold lower sampling frequency due to motion blur (Fig. 4C). Thus, in combination with live cell imaging, the method according to the invention, also enables nanoscale 2D spatial imaging at sub second temporal resolution with minimal photobleaching.
  • Silver particles used for the experiments are from econix silver nanospheres (Nanocomposix) and have a diameter of 50-70 nm and a concentration ⁇ 5.2 x10 12 particles/mL.
  • Camera characterization may be done following the approach of Huang et al. [7] and Diekmann et al. [8], For dark pixel offset, and read noise measurements, it is ensured that the camera chip is in darkness during the acquisition of 2000 frames.
  • the baseline (offset) for each pixel is determined by the mean value per pixel and the read noise by the standard deviation over all frames.
  • Widefield images may be recorded using a VisiTIRF Cell Explorer, a custom modified Nikon (Eclipse Ti-E) inverted microscope with a motorized linear TIRF condenser by Visitron systems.
  • the main objective used in the experiments is a 60XS Oil (CFI Plan Fluor) with iris, NA 1.25 and working distance 0.22 mm. Otherwise, an 100X Oil NA 1.49 (CPI Plan Apo TIRF) objective may be applicable as well. However, due to its short working distance, the suspension may be added on top of the objective directly. To reach 90X magnification with the 60X objective, an intermediate magnification of 1.5X inside the body of the microscope was used.
  • the microscope is equipped with a Perfect Focus System (NIR laser based PFS) and a Hamamatsu Orca Flash4.0 V3 camera with a pixel size of 6.5 pm. Since this is a sCMOS camera with pixel-based statistics, all pixel readouts may be characterized before analysis.
  • NIR laser based PFS Perfect Focus System
  • Flash4.0 V3 camera with a pixel size of 6.5 pm. Since this is a sCMOS camera with pixel-based statistics, all pixel readouts may be characterized before analysis.
  • Double sticky tape was placed on ultra-thin glass slide (thickness: 100 pm) of the same dimensions like the Nunc, Lab-Tek 8-well chambered coverglass system with borosilicate glass bottom 1.0 (#155411, ThermoFisher Scientific). 7 pL of silver nanoparticle suspension (4% silver stock v/v/ in 97% TDE (diluted in ddH2O water)) were added in every well-spot. Before preparing the silver suspension with TDE, silver stock was vigorously vortexed for 60 sec and then mixed thoroughly by pipetting in 97% TDE.
  • PSF Planar Biharmonic Scenor
  • the microscope PSF is measured and estimated for each channel.
  • Tetraspeck beads suspension was added for 1 hour. Afterwards, this suspension was removed and only beads that stick to the surface of the slide remain. From these beads the PSF can be estimated with high accuracy.
  • Fig. 1 Relationship between the low-resolution compressed image (y) (and the superresolution decompressed image (x) .On the left column the schematic image is shown depicting a low-resolution image on the top and a high-resolution decompressed image x where two emitters (white pixels) as ground truth can be seen.
  • the decompressed image comprises s 2 -times more pixels. Imaging (compression) the two emitters results in the low-resolution image, wherein applying the compressed sensing method (decompression) recovers the two emitters.
  • the upper panel describes in more detail the image formation process, where a sensing matrix describes the linear mapping from x to y .
  • a sensing matrix describes the linear mapping from x to y .
  • Z ⁇ modulation matrix
  • the result 102 is convolved with the PSF 103, each s x s pixels are summed up 105 into a new compressed pixel (s: the scale factor). This process is repeated 106 such that a series of low-resolution images is generated 107.
  • Fig. 2 Quantification of the effect of increasing number of frames on reconstruction precision (shown as a grey scale LUT, the lower the grey value the better the separation precision). The absolute difference to the template distance is used to calculate the precision score (%). Mean precision score for the 12 runs in the presence (left graph), or absence (right graph) of random intensity modulation. Black entries in the matrix indicates that no separation took place in the 12 runs.
  • Fig. 3 Comparison of precision scores (%) with 20 frames, in the presence of intensity modulation 300, in the absence of modulation 301 , with standard deconvolution using Huygens software 302 or simple intensity thresholding 303.
  • Fig. 4 Reconstruction of synthetic microtubules 403, 407 with pixel size 36 nm (upper panel) or 24 nm (lower panel.
  • Right reconstructed images Scale bars 75 nm. Intensity units are arbitrary digital units (DN).
  • BEAM Brownian Excitation Absorption Microscopy
  • Fig. 5 Imaging system for compressive imaging.
  • Parallel light 500 coming out of the objective 501 passes through a viscous layer of 97% TDE 503 containing Ag particles 502 of diameter 50-75 nm.
  • the Ag particles 502 undergo Brownian motion in the viscous suspension 503 giving rise to the signal fluctuations in the detected signal.
  • the dark grey shaded areas indicate a strong excitation by the excitation light 500, wherein the brighter areas indicate a lower excitation level, due to the AG particles 502.
  • Fig. 6 Gattaquant STED-90 nm nanoruler reconstruction.
  • STED-90 nm Oregon Green slides were assembled over a layer of Ag suspension and 20 consecutive frames were acquired and used for reconstruction with pixel size 27 nm.
  • Graph shows quantification of distances from 132 reconstructed rulers (Mean ⁇ S.D.).
  • Fig. 7 Reconstruction of real microtubules.
  • COS-7 cells stained for p-tubulin are mounted on a layer of Ag particles containing suspension and 20 consecutive frames were taken and used for reconstruction with pixel size 36 nm.
  • Fig. 8 Schematic representation of the two imaging modalities used to capture mitochondrial dynamic behavior.
  • COS-7 cells transiently expressing EGFP-TOM20 were continuously imaged at a frame rate of 25 fps for 1000 images and a total time of 40 s.
  • the whole sequence may be chopped into blocks of 20 frames and each 20 frames are used to reconstruct one super-resolved image or 10 frames with a moving window of 5 frames maybe used.
  • the images represent a movie reconstruction corresponding to 0,8 s time intervals between each image.
  • Fig. 9 Scheme of dividing original image pixels into s-times oversampled grids with smaller pixel sizes (digital grid).
  • Fig. 10 Schematically depicts a system 1 according to the invention.
  • the system 1 comprises a light source 2, for example a laser, that emits laser light that is guided toward a first objective 3, via a wavelength dependent beam-splitter 14.
  • the light propagates through the first objective 3, which in the present case is an oil immersion objective.
  • the sample carrier 5 is arranged between the objective 3 and the sample 4 comprising a plurality of excitable emitter (not shown).
  • the sample carrier 5 is arranged.
  • the sample carrier 5 is formed by a container comprising a first window 6, a second window 12 that are arranged opposite from each other and extend parallel to each other.
  • the container walls are formed by a wall element 15 that laterally encloses a container volume.
  • the suspension 10 is comprised in which the particles 11 randomly diffuse.
  • the suspension may have the same refractive index as the first and the second window, wherein the windows may be made of glass.
  • the excitation light passes through the suspension, which causes a varying excitation of the emitters at the sample 4.
  • the detected light passes through the beam splitter 14, and is focussed with an imaging lens 7 onto the detector 9 after begin filtered with a filter 8 for residual light that is not within a selected detection wavelength.
  • the detector outputs a signal that comprises information about the emission signal of the emitters. Said signal may be used for reconstructing a series of low-resolution images of the emitters in the sample 4.
  • the computer may be configured to reconstruct a series of high-resolution images from said low-resolution images.
  • sample carrier may be used independently of the other components of the system and the specification of the system.
  • the suspension 10 comprised in the container volume may have a selected viscosity and particles of a selected size distribution.
  • Fig. 11 a similar embodiment of Fig. 10 is shown.
  • the sample carrier 5 essentially consist only of the first window 6.
  • the suspension comprising the particles 10 is provided to the system 1 in form of an immersion medium directly between the objective and the first window.
  • a plot is shown that specifies that number of frames (x-axis) that are required in order to increase the probability of success (gray shading) in resolving an underlying structure having a predefined number of non-zero pixels (y- axis).
  • the density is a measure of how many pixels are non-zero relative to the total number of pixels.
  • the underlying structures for density of 30 (upper row of right panel) and 4 emitters (lower row of right panel) are shown in the plots in the right panel.
  • the underlying structure of emitters is shown as the ground truth (“GT”), and the resulting super-resolved images after processing 4 frames (“SR 4 frames”) and 20 frames (“SR 20 frames”).
  • Fig. 12 B a similar plot of a simulated emitter distribution is shown, wherein a line density of lines that are arranged in a relative spacing of the Full-Width at Half Maximum (FWHM) of the Point Spread Function is shown.
  • FWHM Full-Width at Half Maximum
  • the method according to the invention is able to resolve the underlying emitter distribution.
  • the line density is FWHM/1.5 meaning the lines are arranged below 1.5 times below diffraction limit.
  • the method resolves the two-line structure (upper row) as well as the four-line structure (lower row). For an increased line density of three lines in the PSF (FWHM / 3) the method fails to resolve the four- line structure (lower row) but is capable to resolve the two-line structure (upper row). If the density is increased even further (FWHM /4), the method does not resolve the two- lines nor the four-line structure.

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Abstract

L'invention concerne un système d'imagerie (1) et un procédé d'imagerie à super-résolution de champ lointain microscopique ayant une résolution optique, le système d'imagerie (1) comprenant au moins les composants suivants : - Une source de lumière d'excitation (2) configurée pour fournir une lumière d'excitation à l'intérieur d'une plage de longueurs d'onde d'excitation prédéfinie, - un premier objectif (3) agencé et configuré pour éclairer un échantillon (4) dans un espace d'échantillon (SP) du système d'imagerie (1), l'échantillon (4) comprenant des émetteurs (506) qui sont excitables avec la lumière d'excitation et qui émettent une lumière d'émission lors de l'excitation avec une lumière d'excitation, - un support d'échantillon (5) agencé et configuré pour supporter l'échantillon (4) dans l'espace d'échantillon (SP), le porte-échantillon (5) comprenant une première fenêtre transparente (6) pour supporter l'échantillon (4), le porte-échantillon (5) étant agencé de telle sorte que la lumière d'excitation provenant du premier objectif (3) se propage à travers la première fenêtre (6) du porte-échantillon (6) avant d'atteindre l'espace d'échantillon (SP), - une optique de détection de champ lointain (3, 7) configuré et agencé de telle sorte que, dans le système, une lumière d'émission est collectée à partir de l'espace d'échantillon (SP) et se propage vers un espace d'image le long d'un trajet de détection, - un détecteur (9) disposé dans l'espace d'image et configuré pour enregistrer la lumière d'émission provenant de l'échantillon (4), - une suspension colloïdale (10, 503) comprenant des particules (11, 502), les particules (11, 502) présentant des propriétés de diffusion de la lumière, d'absorption et de plasmonique, la suspension (10, 503) étant disposée dans l'espace d'échantillon (SP) ou entre la première fenêtre (6) du porte-échantillon (5) et le premier objectif (3), de telle sorte que l'excitation des émetteurs (506) avec une lumière d'excitation varie de manière aléatoire à la fois dans l'espace et dans le temps aux positions des émetteurs (506), en raison des propriétés de diffusion, d'absorption et de plasmonique des particules (11, 502) subissant un mouvement brownien dans la suspension (10, 503), de telle sorte que la lumière émise par les émetteurs (506) varie de manière aléatoire.
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