EP3033647A1 - Microscopie de fluorescence 3d haute résolution - Google Patents

Microscopie de fluorescence 3d haute résolution

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Publication number
EP3033647A1
EP3033647A1 EP14739846.5A EP14739846A EP3033647A1 EP 3033647 A1 EP3033647 A1 EP 3033647A1 EP 14739846 A EP14739846 A EP 14739846A EP 3033647 A1 EP3033647 A1 EP 3033647A1
Authority
EP
European Patent Office
Prior art keywords
sample
radiation
image
substance
illumination
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP14739846.5A
Other languages
German (de)
English (en)
Inventor
Thomas Kalkbrenner
Ingo Kleppe
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Carl Zeiss Microscopy GmbH
Original Assignee
Carl Zeiss Microscopy GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Carl Zeiss Microscopy GmbH filed Critical Carl Zeiss Microscopy GmbH
Publication of EP3033647A1 publication Critical patent/EP3033647A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/16—Microscopes adapted for ultraviolet illumination ; Fluorescence microscopes
    • 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
    • G02B21/06—Means for illuminating specimens
    • 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
    • G02B21/00—Microscopes
    • G02B21/36—Microscopes arranged for photographic purposes or projection purposes or digital imaging or video purposes including associated control and data processing arrangements
    • G02B21/365—Control or image processing arrangements for digital or video microscopes
    • G02B21/367—Control or image processing arrangements for digital or video microscopes providing an output produced by processing a plurality of individual source images, e.g. image tiling, montage, composite images, depth sectioning, image comparison
    • G—PHYSICS
    • 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 invention relates to a microscopy method or a microscope for generating a highly resolved in the depth direction image of a fluorescent sample.
  • microscopy methods For resolutions beyond the diffraction limit given by the laws of physics, various approaches have been developed over time. These microscopy methods are characterized in that they provide the user with a higher lateral optical resolution compared to the conventional microscope. In this specification, such microscopy methods are referred to as high-resolution microscopy methods, since they achieve a resolution beyond the optical diffraction limit. Diffraction-limited microscopes, on the other hand, are called classical microscopes. From the publication T. Dertinger et al., "Fast, background-free, 3D super-resolution optica! fluctuation imaging (SOFI) ", PNAS (2009), pp.
  • SOFI super-resolution optica! fluctuation imaging
  • Geissbuehler et al., Biomed., Opt., Express 2, 408-420 (201) discloses a high resolution, far field microscopy method If the fluorophores of a sample flash statistically independently of one another, a sample of the sample can be achieved by suitable filtering with a so-called cumulant function a significant increase in resolution beyond the physically prescribed optical resolution limit
  • a sequence of individual images is recorded and then combined with the cumulant function to a single image, which then di e has higher resolution.
  • This method is referred to by abbreviation of the term "Super-Resoiution Optical Fluctuation Imaging" as the SOFI method.
  • the SOFI method In the SOFI method, a sequence of biases with as many flashes as possible of the fluorophores subsequently added to the sample or inherently present in the sample is required. At the same time, the camera must be able to record this flashing in time and at the same time to offer a high spatial resolution.
  • the SOFI method When realizing the SOFI principle, it must be ensured that as few fluorophores change their fluorescence state during the recording of a single image, and that the fluctuations of individual fluorophores (ie the change in the fluorescence state) can be detected from frame to frame.
  • the SOFI method has therefore been used in the past particularly with regard to thin samples that have virtually no depth extension along the optical axis of the image with respect to the fluorescent material.
  • the invention has for its object to provide a high-resolution microscopy method according to the SOFI principle, with which even thick samples can be analyzed, d. H. the restrictions on the possible samples are lifted.
  • a microscopy method for producing a high-resolution image of a sample comprising the following steps: a) providing the sample with a substance which emits certain fluorescent fluorescence statistically flashing after excitation, or using a sample containing such a substance contains b) irradiation of illumination radiation on the sample and thereby exciting the sample to emit fluorescence radiation.
  • the irradiation of the illumination radiation takes place in such a way that the illumination radiation excites the sample along the optical axis only in a limited depth range for emitting the fluorescence radiation.
  • the SOFI principle is combined with optical cutting methods in order to achieve a high-resolution imaging of the fluorescent sample in the depth direction as well. This avoids both extra-focal background contributions during image generation and also a load on the sample due to fluorescence excitation in depth sections that are not even imaged.
  • the optical cutting can be done in various ways. In one embodiment, a so-called temporal focusing is used, as described for example in DE 102009060793 A1 of the applicant. In another embodiment, a light blue transverse to the optical axis of the image is irradiated. Another embodiment uses multiphoton processes to generate the flashing states in the sample.
  • the sample will fluoresce flatly only in the depth ranges previously selected by the rasterized multiphoton action.
  • the switching radiation is rasterized introduced, preferably with a Mehrphotonenske, since that allows a particularly narrow defined depth range to define.
  • the Umschaitstrahlung can also be introduced by means of temporal focusing, to make the multi-photon excitation without scanning scanning depth-selective.
  • the subsequent excitation of the specimen is carried out without further structuring since the specimen was only switched over in the previously prepared depth ranges and thus can only show the blinking behavior required for the SOFI principle.
  • the aforementioned sample preparation by means of the switching radiation ensures that only a selected depth range shows a specific flashing behavior, which is then evaluated in the SOFI process.
  • one or more of the following variables are possible as blinking parameters: dark time duration, transition probability between dark and bright state of the flashing, light / dark time ratio of the flashing.
  • a flashing parameter of the marker or of the sample can be adjusted by the mentioned illumination parameters, which influences the dark time duration and / or a transition probability between dark and bright state of the BSinking; both with the goal of achieving the optimal ratio of 1: 1 or to approach him.
  • a manipulation of the substance can also be achieved by means of chemical control of a lifetime of the responsible molecules in which fluorescence radiation is emitted (bright state) or no fluorescence radiation is emitted (dark state). In this case, an occupation number of the states is sought, which achieve a transition probability between bright and dark of 0.5 with equal lifetimes of light and dark state.
  • FIG. 1 shows a block diagram of an embodiment of a microscopy method for generating a high-resolution image also in depth orientation
  • FIG. 2 is a schematic representation of a microscope for carrying out the method of FIG.
  • Fig. 3 is a schematic representation of another microscope for performing the method.
  • Fig. 4 is a block diagram similar to Fig. 1 for a further embodiment of the method, which can be carried out with modified constructions of the microscope of Fig. 2 or 3.
  • Figure 1 shows as a block diagram of a first embodiment of a microscopy method for generating a high-resolution image in the depth direction.
  • a sample is provided with a marker, which eats the substance mentioned in the beginning, which emits statistically flashing certain fluorescence radiation after excitation.
  • a sample is selected which already contains the substance.
  • a subsequent step S2 illumination radiation is irradiated onto the sample and thus the emission of the determined fluorescence radiation from the substance in the sample is excited.
  • the illumination radiation is irradiated by means of an optical cutting method so that it excites the sample along the optical axis of a subsequent image only in a limited depth range for emitting the fluorescence radiation. This limited depth range determines the resolution in the depth direction.
  • step S3 the sample is repeatedly imaged in a step S3, wherein due to the flashing behavior in each image, a different flashing state of the sample is present.
  • the repeated image thus generates a sequence of images In.
  • this image sequence In is processed by means of a cumulant function, which evaluates intensity fluctuations caused by the flashing in the image sequence.
  • an image If is generated that has a spatial resolution that exceeds the optical resolution of the image.
  • the method of steps S3 and S4 corresponds to the known SOFI principle, for example according to the publication by Dertinger et al. The difference, however, is that due to the embodiment of step S2, the sample emits flashing fluorescence radiation only in a narrowly defined depth range.
  • FIG. 2 shows a microscope 1 which can be used to carry out the method of FIG.
  • FIG. 2 two different embodiments for the microscopy method are entered.
  • the elements 17 to 19 and the punctured beam path in the image of Figure 2 do not affect the embodiment of the method according to Figure 1. These components of Figure 2 will therefore be explained later and should not play any role for the time being.
  • a sample 2 is located behind a cover glass not specified. It is imaged with the microscope 1 via an objective 3 and a tube lens 4 onto a detector 5. This corresponds to a known microscope structure.
  • a beam splitter 7 via which an illumination beam path 8 is coupled, which has a beam shaping device 1 1, which introduces the radiation via the microscope 3 in the sample 2.
  • the illumination beam path 8 comprises an illumination source 9, which emits the illumination radiation 10.
  • the illumination radiation 10 is pulsed and is irradiated by temporal focusing so that it has a certain pulsed time behavior only in a limited depth range. Only in a limited depth range of the sample, the pulse duration is minimized.
  • the illumination source 9 outputs the pulsed illumination radiation 10. It is deflected via a scattering element, which is formed in the embodiment shown in Figure 2 as a grid 12. Instead of the grid 12, other scattering elements can be used, for. As a DMD, LDC filter, LCoS or a dispersive element.
  • a scattering element which is formed in the embodiment shown in Figure 2 as a grid 12.
  • other scattering elements can be used, for.
  • a DMD LDC filter, LCoS or a dispersive element.
  • the illumination source 9 thus emits a pulsed raw beam, which is modified via the scattering element and the optics so that only in the image plane 15, which lies in the sample 2, the minimum pulse length is given after the scattering element. Above and below the image plane 15, the pulse length is greater.
  • the beam path of the illumination beam path 8 is shown drawn through.
  • the radiation from one element of the grating 12 is shown in dashed lines.
  • the radiation incident on the grating elements of the grating 12 is spectrally decomposed.
  • the spectral components of the radiation have the same running time only for the image plane 5, so that only in the image plane 15 the pulses of the raw beam, as it comes from the illumination source 9, are reconstructed into pulses with a minimum pulse length. This applies in the entire image plane 15, as the solid illumination beam path shows.
  • FIG. 3 shows a further embodiment of the method of FIG. 1 in the form of a schematically illustrated microscope 1. Elements that correspond functionally or structurally to those of FIG. 2 have the same reference numerals in order to dispense with repeated descriptions.
  • the microscope 1 of Figure 3 differs from the microscope of Figure 2 essentially by the illumination beam path 8.
  • the illumination source 9 is here from a light beam, which is also modified by a beam shaping device 11. 2
  • the beam shaping device 1 was still formed by the grating 12 and the optics 13 and 14 for optical cutting by temporal focusing
  • the beam shaping device 11 of FIG. 3 effects the irradiation of the illumination radiation in the form of a light sheet 16 transverse to the optical axis 6 of the microscope 1
  • the sample 2 is thus irradiated only in the region of the light sheet 16, which consequently determines the depth plane.
  • FIG. 4 schematically shows a further embodiment of the microscopy method.
  • steps S3 and S4 it corresponds to that of FIG. 1, so that the repetition of the description of these steps can be dispensed with.
  • the differences lie in the embodiment of step S2, which is formed in two parts in the embodiment of FIG. It consists of two steps S2a and S2b.
  • the step S1 is modified to a step S1 '.
  • a sample is provided whose substance is separated by a Multiphoton effect is switched to a fluorescence state in which it emits the SOFi-compatible fluorescence radiation with the statistical Bünk . In other words, it is only after switching on a switching radiation that the sample (ie its fluorescent molecules) can be excited.
  • the step SV therefore comprises the marking of a sample with a substance or the selection of a sample inherently suitable substances, which can be set by means of Umschaitstrahlung in a state in which it can then be excited by irradiation of an excitation radiation for emitting fluorescence radiation.
  • the illumination radiation which was irradiated in step S2 of the embodiment of FIG. 1 thus comprises in the embodiment of FIG. 4 two components, a switching radiation and an excitation radiation. Accordingly, the step S2 is divided into two steps S2a and S2b.
  • step S2a the switching radiation is radiated. This is done so that the desired depth range is selected.
  • step S2b the excitation radiation is then irradiated onto the sample. In this case, depth depth selection no longer has to take place, since the sample can only emit fluorescence radiation in those areas which were previously switched over by the irradiation in step S2a.
  • the bipartite provision of the illumination radiation has a significant advantage.
  • the sample is scanned, if in step SV a corresponding substance was used, which is switched over a multi-photon effect.
  • step S2a temporal focusing may be considered in step S2a.
  • the microscope of Figure 2 is therefore structured for an alternative embodiment of the method in contrast to the previously described structure so that the illumination source 9 provides the switching radiation pulsed.
  • the pulse length and thus the intensity required for the multiphoton process is present exclusively in the image plane 15.
  • a beam splitter 17 is additionally provided, which couples light from a beam source, which then acts as excitation radiation 19, into the beam path of the microscope 1, the sample being illuminated in the wide field. Only the previously prepared areas in the image plane 15 then emit the statistically flashing fluorescence radiation.
  • the illumination radiation is realized by the combination of the illumination beam path 8 and the excitation beam path ⁇ realized by the elements 17 to 19).
  • Scanning Umschaltstrahiung is not mandatory in this embodiment, since the temporal focus already realizes the depth selection.
  • FIG. 3 shows a microscope for the embodiment of the method according to FIG. 4 - here for a scanned sample preparation by multiphoton process.
  • the elements 9 to 1 1 are modified (not shown) so that they cause a far-field illumination of the sample 2 with excitation radiation.
  • This excitation can take place transversely to the optical axis 6 but alternatively also along the optical axis 6.
  • a beam splitter 20 is provided, which is fed with radiation from a scanner 21, which deflects a raw beam from a switching beam source 22 by scanning.
  • a switching beam 23 which is scanned over the sample 2. It causes by means of Mehrphotonen bin switching the substance sample 2 in a state in which it can then excite the excitation radiation.
  • step S2a is thus carried out by suitable control of the scanner 21 and the switching beam source 22, the step S2b by suitable control of the beam source.
  • a (not shown) control device which controls the components of the microscope for carrying out the method of Figure 1 or Figure 4 suitable.
  • the image sequence In from individual images is converted into the high-resolution image If in the SOFI processing S4.
  • SOFI processing S4 for example, by Dertinger et al. described principle used. Likewise, the principle of Dertinger et al. Further developed concept according to WO 2010/141608 A1 can be used. This publication is also fully included in this regard.
  • the florescence of the fluorophores required for the SOFI principle is defined by a transition from a first, fluorescent to a second, non-fluorescent state.
  • a non-fluorescent state is understood to be any state in which the fluorescence radiation which is evaluated for the image is not emitted.
  • the non-fluorescent state can thus definitely be a state in which a fluorophore shines in another fluorescence spectral range.
  • transition probabilities from the first to the second state can be modified, as is known, for example, from the publication Heilemann et al., Angewandte Chemie 121, p. 7036, 2009, for example by chemical influences, temperature influences or lighting influences.
  • the SOFI principle is particularly efficient when the proportion between luminous and non-luminous fluorophores for the respective image acquisition or image integration time is 1: 1.
  • the transition probability between the first and second states and between the second and first states should ideally be 0.5. This can be achieved by appropriate manipulation of the sample by means of chemical action, temperature action or illumination. In this case, by optimizing the incident spectral distribution, the transition probability can be optimized with the aim of achieving the mentioned opiate ratio 1: 1.
  • the SOFI principle therefore requires transition probabilities that are significantly different from other microscopy techniques.
  • the PALM principle also referred to as dSTORM
  • dSTORM calls for states where the vast majority of fluorophores are in a dark state.
  • the dark time period must be taken into account in addition to the transition probability. Even if the transition probability from light to dark is 0.5, with a much longer life of the dark states, the optimal ratio of 1: 1 would be missed.
  • the means for modifying the transition probability and dark time duration used in the prior art are therefore particularly preferably used (and regardless of the imaging of an image field which can be smaller than a sample field), the ratio of luminous to non-luminous fluorophores in the direction Optimize 1: 1 optimally by adjusting transition probability and / or dark life (or helix duration) appropriately and adjusting to the image acquisition rate or integration time. Conversely, it is possible to adapt the absorption rate to the lifetimes.

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  • Physics & Mathematics (AREA)
  • General Physics & Mathematics (AREA)
  • Optics & Photonics (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Multimedia (AREA)
  • Computer Vision & Pattern Recognition (AREA)
  • Health & Medical Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Microscoopes, Condenser (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
  • Analysing Materials By The Use Of Radiation (AREA)

Abstract

L'invention concerne un procédé de microscopie permettant de produire une image haute résolution (/f) d'un échantillon (2) et comprenant les étapes suivantes consistant à : a) munir l'échantillon (2) d'une substance qui émet après excitation un rayonnement fluorescent déterminé clignotant statistiquement, ou utiliser un échantillon (2) contenant ladite substance ; b) émettre un rayonnement lumineux (10) sur l'échantillon (2) et exciter ainsi l'échantillon (2) pour qu'il émette le rayonnement fluorescent ; c) reproduire à répétition l'échantillon (2) émettant le rayonnement fluorescent le long d'un axe optique (OA) sur un détecteur à résolution locale (5), de sorte que l'on obtient une succession d'images (In) ; d) traiter la succession d'images (In) au moyen d'une fonction cumulative qui évalue les fluctuations d'intensité provoquées dans l'image par le clignotement, et produire ainsi une image (If) qui présente une répartition locale de la substance dans l'échantillon (2) dont la résolution locale a augmenté au-delà de la résolution optique de la reproduction, e) l'émission du rayonnement lumineux (10) étant effectuée de telle manière que le rayonnement lumineux (10) n'excite l'échantillon (2) le long de l'axe optique (OA) que dans une plage de profondeur limitée pour l'émission du rayonnement fluorescent.
EP14739846.5A 2013-08-14 2014-07-18 Microscopie de fluorescence 3d haute résolution Withdrawn EP3033647A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE201310216124 DE102013216124A1 (de) 2013-08-14 2013-08-14 Hochauflösende 3D-Fluoreszenzmikroskopie
PCT/EP2014/065501 WO2015022146A1 (fr) 2013-08-14 2014-07-18 Microscopie de fluorescence 3d haute résolution

Publications (1)

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EP3033647A1 true EP3033647A1 (fr) 2016-06-22

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EP14739846.5A Withdrawn EP3033647A1 (fr) 2013-08-14 2014-07-18 Microscopie de fluorescence 3d haute résolution

Country Status (6)

Country Link
US (1) US20160195704A1 (fr)
EP (1) EP3033647A1 (fr)
JP (1) JP2016534395A (fr)
CN (1) CN105452931B (fr)
DE (1) DE102013216124A1 (fr)
WO (1) WO2015022146A1 (fr)

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP6507582B2 (ja) * 2014-11-13 2019-05-08 セイコーエプソン株式会社 印刷装置
DE102015121920A1 (de) 2015-12-16 2017-06-22 Carl Zeiss Microscopy Gmbh Hochauflösendes Kurzzeit-Mikroskopieverfahren und hochauflösendes Kurzzeit-Mikroskop
US10901230B2 (en) * 2016-06-21 2021-01-26 Illumina, Inc. Super-resolution microscopy
DE102017115658A1 (de) * 2017-07-12 2019-01-17 Carl Zeiss Microscopy Gmbh Flackern bei Winkel-variabler Beleuchtung
PL3623798T3 (pl) * 2018-09-13 2022-03-28 Euroimmun Medizinische Labordiagnostika Ag Sposób i urządzenie do wykrywania i przedstawiania obrazu immunofluorescencyjnego próbki biologicznej
DE102018215831B4 (de) * 2018-09-18 2020-04-02 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Optische Anordnung für fluoreszenzmikroskopische Anwendungen
DE102018128590A1 (de) 2018-11-14 2020-05-14 Carl Zeiss Microscopy Gmbh Fluktuationsbasierte Fluoreszenzmikroskopie

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102008009216A1 (de) * 2008-02-13 2009-08-20 Carl Zeiss Microimaging Gmbh Vorrichtung und Verfahren zum räumlich hochauflösenden Abbilden einer Struktur einer Probe

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7408636B2 (en) * 2002-10-31 2008-08-05 Chemimage Corporation Method and apparatus for dark field chemical imaging
CN1953701A (zh) * 2004-03-11 2007-04-25 通用医院有限公司 使用荧光蛋白进行断层摄影成像的方法和系统
US8625863B2 (en) 2009-06-02 2014-01-07 Sofast Gmbh Superresolution optical fluctuation imaging (SOFI)
DE102009060079A1 (de) 2009-11-19 2011-05-26 Liebherr-Hausgeräte Ochsenhausen GmbH Kühl- und/oder Gefriergerät
DE102009060793A1 (de) * 2009-12-22 2011-07-28 Carl Zeiss Microlmaging GmbH, 07745 Hochauflösendes Mikroskop und Verfahren zur zwei- oder dreidimensionalen Positionsbestimmung von Objekten
DE102010044013A1 (de) * 2010-11-16 2012-05-16 Carl Zeiss Microimaging Gmbh Tiefenauflösungsgesteigerte Mikroskopie
EP2535755A1 (fr) * 2011-06-14 2012-12-19 Ecole Polytechnique Fédérale de Lausanne (EPFL) Microscopie de cumulant
US9395302B2 (en) * 2012-07-25 2016-07-19 Theranos, Inc. Image analysis and measurement of biological samples

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102008009216A1 (de) * 2008-02-13 2009-08-20 Carl Zeiss Microimaging Gmbh Vorrichtung und Verfahren zum räumlich hochauflösenden Abbilden einer Struktur einer Probe

Also Published As

Publication number Publication date
US20160195704A1 (en) 2016-07-07
WO2015022146A1 (fr) 2015-02-19
JP2016534395A (ja) 2016-11-04
DE102013216124A1 (de) 2015-02-19
CN105452931B (zh) 2018-09-11
CN105452931A (zh) 2016-03-30

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