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EP-4573359-B1 - METHOD AND DEVICE FOR SIMULTANEOUSLY TRACKING TWO EMITTERS

EP4573359B1EP 4573359 B1EP4573359 B1EP 4573359B1EP-4573359-B1

Inventors

  • DONNERT, GERALD
  • REUSS, MATTHIAS
  • FISCHER, JOACHIM
  • SCHMIDT, ROMAN
  • Engelhardt, Tobias
  • WILLEMER, WINFRIED

Dates

Publication Date
20260506
Application Date
20230818

Claims (15)

  1. A method for simultaneously tracking the movements of a first isolated emitter and a second isolated emitter optically distinguishable from the first emitter by means of a MINFLUX method or a STED-MINFLUX method, wherein the first and second emitters are spaced apart such that, when a MINFLUX method is performed on one of the emitters, the other emitter necessarily enters the influence range of the excitation light, or when a STED-MINFLUX method is performed on one of the emitters, the other emitter necessarily enters the influence range of the emission suppression light, in a sample comprising: (a) an illumination and measurement step in which the sample is illuminated with intensity distributions of excitation light or of emission-suppressing light overlapping with excitation light, which comprise a local minimum, in particular a zero point, within a measurement range, and intensity increase regions adjacent to the minimum, wherein, in a temporal sequence, various profiles of intensity increase regions from a predetermined set of profiles of intensity increase regions are generated in a presumed location region of at least the first emitter and, optionally, of profiles of intensity increase regions in a presumed location region of the second emitter, and emissions from the first emitter and the second emitter are measured, wherein measured emission values are assigned to the respective profiles of intensity increase regions, and b) a location determination step, in which, on the basis of the various profiles of intensity increase regions and the assigned emission measurement values, a determination is made of either i) a location of the first emitter, or ii) locations of the first and second emitters, or iii) a common average location of both emitters, c) wherein, based on i) the location of the first emitter determined in the location determination step, or ii) the locations of both emitters determined in the location determination step, or iii) the common average location of both emitters determined in the location determination step, a respective new presumed location region is determined for each of the emitters and a new set of profiles is determined in total, d) wherein the illumination and measurement step and the location determination step are repeated using the new set of profiles of intensity increase regions, and e) wherein the excitation light of the illumination and measurement step comprises first excitation light for exciting the first emitter and second excitation light for exciting the second emitter.
  2. A method according to claim 1, characterised in that the first and second excitation lights differ in terms of their spectral composition, in particular wherein, in the illumination and measurement step, illumination with the first excitation light and illumination with the second excitation light take place at different times.
  3. A method according to any one of the preceding claims, characterised in that the local minimum is a 3D minimum, or that the local minimum is a 2D minimum, or that the local minimum is a 1D minimum, in particular wherein, in the illumination and measurement step, minima of different types and/or minima with different orientations are used alternately, and/or that the illumination and measurement step is carried out and repeated using minima of different types and/or minima with different orientations alternately.
  4. A method according to one of the preceding claims, characterised in that the excitation light is pulsed, in particular wherein the measurement of the emission excited by a respective pulse is performed in a time-resolved manner, further in particular with a time resolution better than 1 ns, further preferably better than 100 ps, and even more preferably better than 30 ps.
  5. A method according to any of the preceding claims, characterised in that , in the location determination step, the locations of the first and second emitters are determined on the basis of the various profiles and the associated measured values of the emission, and that, on the basis of the locations of both emitters determined in the location determination step, a respective new presumed location region is determined for each of the emitters and, in total, a new set of profiles is determined.
  6. A method according to claim 5, characterised in that the predetermined set of profiles comprises a first predetermined subset of profiles adapted to a presumed location region of the first emitter, and a second predetermined subset of profiles adapted to a supeceted location region of the second emitter, in particular wherein the first predetermined subset of profiles and the second predetermined subset of profiles differ from one another, and further in particular wherein, in the illumination and measurement step, the profiles of the first excitation light or the profiles of the emission suppression light, which overlaps with the first excitation light, are in accordance with the first predetermined subset of profiles, and the profiles of the second excitation light or profiles of an emission suppression light that overlaps with the second excitation light are generated in accordance with the second predetermined subset of profiles.
  7. The method according to claim 6, characterised in that , in the illumination and measurement step, the profiles of the first excitation light or the profiles of the emission suppression light, which overlaps with the excitation light, are generated in accordance with the first predetermined subset of profiles, and subsequently the profiles of the second excitation light or of an emission suppression light that overlaps with the second excitation light are generated in accordance with the second predetermined subset of profiles, wherein, optionally, after the profiles of the first excitation light or of the emission suppression light, which overlaps with the first excitation light, have been generated in accordance with the first predetermined subset of profiles, within a period during which the illumination and measurement step is continued with the second excitation light, the location determination step is initiated based on the various profiles of the first predetermined subset of profiles and the associated emission measurement values, wherein the location of the first emitter is determined, and, based on the location of the first emitter determined in the location determination step, a new presumed location region and a new first subset of the new set of profiles are determined for the first emitter, wherein the new first subset of profiles comprises profiles adapted to the new presumed location region of the first emitter, and/or after the profiles of the second excitation light or the profiles of the emission suppression light, which overlaps with the second excitation light, have been generated in accordance with the second predetermined subset, within a period in which the repetition of the illumination and measurement step with the first excitation light is commenced and preferably completed, the location determination step is completed on the basis of the various profiles of the second predetermined subset of profiles and the associated emission measurement values, whereby the location of the second emitter is determined, and, based on the location of the second emitter determined in the location determination step, a new presumed location region and a new second subset of the new set of profiles are determined for the second emitter, wherein the new second subset comprises profiles adapted to the new presumed location region of the second emitter.
  8. A method according to any one of claims 1 to 4, characterised in that , in the location determination step, the location of the first emitter is determined on the basis of the various profiles and the associated measured values of the emission, and in that , on the basis of the location of the first emitter determined in the location determination step, a respective new presumed location region is determined for the first emitter and for the second emitter, and a new set of profiles is determined in total, wherein, optionally, the first and second emitters are connected to one another by a structure, in particular a biological structure, to which they are coupled as markers, wherein, furthermore, in particular, the new presumed location region for the second emitter is determined on the basis of the location of the first emitter determined in the location determination step and on the basis of prior knowledge regarding the structure to which the emitters are coupled.
  9. A method according to any one of claims 1 to 4, characterised in that , in the location determination step, a common average location of both emitters is determined on the basis of the various profiles and the associated emission measurement values, and in that , on the basis of the common average location of both emitters determined in the location determination step, a respective new presumed location region and, in total, a new set of profiles are determined, in particular wherein the first and second emitters are spaced apart by a distance of less than 100 nm or less than 50 nm or less than 20 nm or less than 10 nm or less than 5 nm, wherein, optionally, the common average location of both emitters is determined on the basis of the totality of the measured values of the emission of the first emitter and the second emitter from the illumination and measurement step.
  10. A method according to any one of the preceding claims, characterised in that the various profiles of intensity increase regions are obtained by placing a minimum of an intensity distribution at different positions, such that a set of profiles corresponds to a set of positions of the minimum, in particular wherein the set of positions comprises a first and a second position which are separated by a distance that is at least four times, ten times or fifty times the distance between the two emitters, or at least 50 nm, or at least 100 nm, or at least 200 nm.
  11. A method according to any one of the preceding claims, characterised in that the first and/or the second emitter is a fluorescent emitter, in particular wherein the first and/or the second fluorescent emitter is a fluorophore or a fluorescent unit formed from fluorophores, or characterised in that the first and/or the second emitter is a scattering emitter.
  12. A method according to any one of the preceding claims, characterised in that the spectral compositions of the first excitation light and the second excitation light and the first and second emitters are matched such that excitation of the second emitter by the first excitation light is minimal in relation to excitation of the first emitter by the first excitation light, or, at equal excitation intensity, amounts to at most 10%, preferably at most 5%, more preferably at most 1%, and even more preferably at most 0.1% of the excitation of the first emitter, and/or that the spectral compositions of the first excitation light and the second excitation light and the first and second emitters are matched such that excitation of the first emitter by the second excitation light is minimal or, in particular, at most 5% at equal excitation intensities, preferably at most 1%, more preferably at most 0.1% of the excitation of the second emitter.
  13. A method according to any of the preceding claims, characterised in that the measurement of the fluorescence emission is carried out in two detection channels which differ in terms of spectral sensitivities, in particular wherein the spectral sensitivities of the detection channels and of the first and second emitters are matched such that the sensitivity of the first detection channel to the emission of the second emitter is minimal or at most 10%, preferably at most 5%, more preferably at most 1% of the sensitivity to the emission of the first emitter, , and/or that the spectral sensitivities of the detection channels and the first and second emitters are matched such that the sensitivity of the second detection channel to the emission of the first emitter is minimal or at most 10%, preferably at most 5%, and more preferably at most 1%, of the sensitivity to the emission of the second emitter.
  14. A method according to any of the preceding claims, characterised in that , following the completion of tracking the movement of the first and second emitters, in an evaluation step based on the measured values of the emission and the associated respective profiles obtained in the repeatedly performed illumination and measurement steps, the trajectories of the first and second emitters are determined, optionally taking into account, when determining the trajectories, a distance-dependent influence of the proximity of one emitter on the emission of the other emitter, if present, in particular a Förster resonance energy transfer.
  15. Apparatus configured to carry out a method according to one of the preceding claims for simultaneously tracking the movements of a first isolated emitter and a second isolated emitter optically distinguishable from the first emitter by means of a MINFLUX method or a STED-MINFLUX method, wherein the first and second emitters are spaced apart such that, when a MINFLUX method is performed on one of the emitters, the other emitter necessarily enters the influence range of the excitation light, or when a STED-MINFLUX method is performed on one of the emitters, the other emitter necessarily enters the influence range of the emission-suppression light, in a sample comprising - a light source configured to generate excitation light and, optionally, emission-suppression light, wherein the excitation light comprises first excitation light for exciting the first emitter and second excitation light for exciting the second emitter. - an illumination device configured to control light from the light source such that the excitation light or emission suppression light directed into the sample forms, in overlap with excitation light within the sample in a measurement region, an intensity distribution with a local minimum, - a light control device, in particular a displacement device, which is configured to generate different profiles of intensity increase regions adjacent to the minimum in the sample, in particular to displace the local minimum relative to the sample, wherein the light control device may be an integral part of the illumination device, and wherein, in a temporal sequence, various profiles of intensity increase regions of a predetermined or newly determined set of profiles of intensity increase regions are generated in a presumed location region of at least the first emitter and, optionally, of profiles of intensity increase regions in a presumed location region of the second emitter, - a measuring device configured to detect emissions from the first emitter and the second emitter within the measuring range, wherein measured values of the emission are assigned to the respective profiles of intensity increase regions, - a storage unit configured to store positions of the local minimum and associated measured values, - an evaluation unit configured to evaluate measured values associated with the profiles of intensity increase regions or positions of the local minimum, wherein, on the basis of the various profiles or positions and the associated measured values of the fluorescence emission, either i) a location of the first emitter or ii) locations of the first and second emitters or iii) a common average location of both emitters is determined, and on the basis of i) the determined location of the first emitter, or ii) the determined locations of both emitters, or iii) the determined common average location of both emitters, a respective new presumed location region is determined for each of the emitters, and based thereon a set of profiles of intensity increase regions or of positions of the local minimum is determined, - a control unit configured to control the light control device, wherein, in accordance with the set of profiles, different profiles of intensity increase regions are generated in the sample, in particular wherein the local minimum is shifted to the positions of the determined set of positions.

Description

Technisches Gebiet der Erfindung Die Erfindung betrifft das Gebiet der höchstauflösenden lichtoptischen Mikroskopie. Sie betrifft konkret Verfahren und Vorrichtungen zum simultanen Verfolgen der Bewegungen mehrerer Emitter, beispielsweise fluoreszenzmarkierter biologischer Strukturen, die zueinander während des Verfolgens einen Abstand aufweisen der kleiner ist als Beugungslimit, mit hoher Zeitauflösung. Stand der Technik Als ein Mikroskopieverfahren zum Verfolgen schnell bewegter Partikel ist die interferometric scattering (iSCAT) microscopy bekannt. In der Publikation "Interferometric scattering microscopy reveals microsecond nanoscopic protein motion on a live cell membrane" (Taylor, R.W. et al., Nat. Photonics 13, 480-487 (2019). https://doi.org/10.1038/s41566-019-0414-6) wird eine Anwendung der iSCAT-Mikroskopie auf das Verfolgen von Proteinen in Membranen lebender Zellen beschrieben. Mit dem Verfahren werden bei einer zeitlichen Auflösung von besser als 20 µs örtliche Auflösung besser als 10 nm erreicht. Bei dem Verfahren wird die Interferenz zwischen Referenzlicht und Licht, das an einem an den zu verfolgenden Partikel, beispielsweise einem Biomolekül, gebundenen Marker, beispielsweise einem Gold-Nanopartikel gestreut wird, beobachtet. Das Verfahren ermöglicht zwar eine sehr gute zeitlich-örtliche Auflösung, weist aber den Nachteil auf, dass die zu verwendenden Marker vergleichsweise groß sind und daher großen Einfluss auf die Eigenschaften der Probe ausüben. Das Verfahren ist schon wegen der Größe der Marker nicht geeignet, zwei nur um wenige Nanometer voneinander beabstandete Objekte separat zu beobachten, um deren Bewegungen zu verfolgen. Im Stand der Technik sind kamerabasierte lokalisationsmikroskopische Verfahren der Fluoreszenzmikroskopie bekannt, die eine Fluoreszenzbildgebung mit hoher Ortsauflösung im Bereich zumeist einiger zehn Nanometer in mehreren Farben, das heißt unter Nutzung von Fluorophoren, die stark unterschiedliche Anregungsspektren oder unterschiedliche Emissionsspektren oder beides aufweisen. Die örtlich zeitliche Auflösung ist bei diesen Verfahren durch die Kamera sowie durch die Helligkeit der Emitter limitiert, sodass sie allenfalls für das Beobachten langsamer Bewegungen geeignet sind. Geeignete Emitter sind insbesondere funktionalisierte Core-Shell Quantum Dots, die zwar sehr photostabil und sehr hell sind, aber bislang in der Fluoreszenzmikroskopie eher selten zur Anwendung kommen. Ein solches Verfahren ist beispielsweise in der Publikation "Differential Labeling of Myosin V Heads with Quantum Dots Allows Direct Visualization of Hand-Over-Hand Processivity" (Warshaw DM et al., Biophys J. 2005 May;88(5):L30-2. doi: 10.1529/biophysj.105.061903. Epub 2005 Mar 11. PMID: 15764654; PMCID: PMC1305523.) beschrieben. Es wird eine örtliche Auflösung von 6 nm bei einer zeitlichen Auflösung von 83 ms erreicht. Um die Bewegung von zweifach gefärbten Motorproteinen und damit die Bewegung der Enden der Motorproteine separat beobachten zu können, wurde eine sehr geringe ATP-Konzentration in der Probe eingestellt. Ähnliche Untersuchungen unter Nutzung von in der Fluoreszenzmikroskopie üblicheren Fluorophoren, nämlich Cy3 und Cy5, bei Nutzung zweier Anregungswellenlängen bei einer die Hintergrundfluoreszenz minimierenden TIRF-Beleuchtung werden in der Publikation "Single molecule high-resolution colocalization of Cy3 and Cy5 attached to macromolecules measures intramolecular distances through time" (L. Stirling Churchman et al., Proc Natl Acad Sci U S A. 2005 Feb 1;102(5):1419-23. Doi: 10.1073/pnas.0409487102. Epub 2005 Jan 24. PMID: 15668396; PMCID: PMC545495.) beschrieben. Hier wird bei einer Integrationszeit von 0,5 s eine laterale Auflösung von etwa 6 nm erreicht. Ein Mehrfarben-Scanning-Mikroskop bzw. ein entsprechendes Verfahren, das eine Fluoreszenzbildgebung mit hoher Ortsauflösung im Bereich weniger Nanometer erlaubt, ist aus der Publikation "Ultrahigh-resolution multicolor colocalization of single fluorescent probes" (Thilo D. Lacoste et al., Proc Natl Acad Sci U S A. 2000 Aug 15;97(17):9461-6. Doi: 10.1073/pnas.170286097. PMID: 10931959; PMCID: PMC16886.) bekannt. Hierbei werden verschiedene Fluorophore, die alle mit derselben Wellenlänge angeregt werden, genutzt, da eine bei Nutzung mehrerer Anregungswellenlängen mit Blick auf die zu erreichende Auflösung notwendige Präzision der Überlagerung der Anregungslichtstrahlen nicht möglich sei. Die Probe wird konfokal abgetastet, das Fluoreszenzlicht wird spektral in zwei oder mehr Kanäle aufgespalten, aus den Messwerten wird ein Mehrfarbenbild erzeugt. Mit dem Verfahren werden Abstände zwischen verschiedenfarbigen Fluorophoren im Bereich von etwa 10 nm bestimmt. Dieses Verfahren ist ebenso wenig für das schnelle simultane und separate Verfolgen mehrerer Emitter geeignet wie die hier genannten kamerabasierten lokalisationsmikroskopischen Verfahren. In der Publikation "Nanometer resolution imaging and tracking of fluorescent molecules