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DE-102024133173-A1 - METHOD, DEVICE, LOCATION MICROSCOPE AND COMPUTER PROGRAM FOR LOCATION OF EMISSIONERS IN A SAMPLE

DE102024133173A1DE 102024133173 A1DE102024133173 A1DE 102024133173A1DE-102024133173-A1

Abstract

The invention relates to a method for localizing emitters (E) in a sample (2), wherein the sample (2) is illuminated with structured illumination light (B) to localize an emitter (E), wherein after at least one localization of an emitter (E), it is checked whether an accuracy criterion is met, wherein the accuracy criterion indicates whether the position of the emitter (E) is known with a predetermined expected accuracy, wherein a check is carried out using the position of the emitter (E) determined in the localization if the accuracy criterion is met, wherein during the check information about the emitter (E) or the environment of the emitter (E) is collected, wherein the information is taken into account in at least one evaluation step following the localization or in at least one further localization of an emitter (E), as well as a device, a localization microscope (1) and a computer program for carrying out the method.

Inventors

  • Jörn Heine-von Riegen

Assignees

  • ABBERIOR INSTRUMENTS GMBH

Dates

Publication Date
20260513
Application Date
20241113

Claims (20)

  1. Method for localizing emitters (E) in a sample (2), wherein the sample (2) is illuminated with structured illumination light (B) to localize an emitter (E), wherein after at least one localization of the emitter (E) it is checked whether an accuracy criterion is met, wherein the accuracy criterion specifies whether the position of the emitter (E) is known with a predetermined expected accuracy, wherein a check is carried out using the position of the emitter (E) determined in the localization if the accuracy criterion is met, wherein during the check information about the emitter (E) or the environment of the emitter (E) is collected, and wherein the information is taken into account in at least one evaluation step following the localization or in at least one further localization of an emitter (E).
  2. Procedure according to Claim 1 characterized in that a control variable is determined during the control process.
  3. Procedure according to Claim 2 , characterized in that the control variable is used to determine whether detected light emissions (L) originate from a single emitter (E) or from multiple emitters (E).
  4. Procedure according to Claim 3 , characterized in that the localization of the emitter (E) is discarded if it is determined that the light emissions (L) originate from multiple emitters (E).
  5. Procedure according to Claim 2 , characterized in that the control variable includes information about background light in an environment of the emitter (E).
  6. Procedure according to Claim 2 , characterized in that the control variable includes information about an assignment of the emitter (E) to one of several emitter species.
  7. Procedure according to Claim 2 , characterized in that the control variable includes information about a state, in particular a photophysical or photochemical state, of the emitter (E).
  8. Method according to one of the preceding claims, characterized in that the illumination light (B) is excitation light which excites emitters (E) in the sample (2) to luminescence, in particular fluorescence, or is scattered or reflected by emitters (E) in the sample (2).
  9. A method according to one of the preceding claims, characterized in that the sample (2) is illuminated in a region of a presumed position (V) of the emitter (E) with an intensity distribution of the illumination light (B), wherein the intensity distribution has a local minimum, wherein the local minimum of the intensity distribution is arranged at illumination positions (21) of an illumination pattern (20) around the presumed position (V), wherein light emissions from the sample (2) are detected for each of the illumination positions (21), and wherein the emitter (E) is localized with increased accuracy compared to the presumed position (V) based on the illumination positions (21) and the associated light emissions (L).
  10. Procedure according to Claim 9 , characterized in that localizations of the emitter (E) are obtained in several iteration steps, wherein the iteration steps are carried out without control up to a predetermined iteration step, wherein after carrying out the predetermined iteration step the accuracy criterion is met, and wherein the control is subsequently carried out.
  11. Procedure according to Claim 9 or 10 , insofar as it relates back to one of the Claims 2 until 7 , characterized in that the control variable is a CFR value or is determined on the basis of a CFR value, wherein the CFR value is based on the formula CFR = C F R = p 0 ⋅ ∑ j = 1 m t j t 0 ⋅ ∑ j = 1 m p j is determined, where p 0 denotes a number of detected light emissions (L) at a positioning of the local minimum of the intensity distribution at a control position (22) corresponding to an estimated position of the emitter (E), p j denotes a number of detected light emissions at a positioning of the local minimum of the intensity distribution at an illumination position (21) spaced apart from the control position (22) with the index j, where t 0 denotes a residence time of the local minimum at the control position (22), t j denotes a residence time of the local minimum at the illumination position (21) with the index j, and m denotes the number of illumination positions (21).
  12. Procedure according to Claim 11 , characterized in that the illumination positions (21) used to determine the CFR value form an illumination pattern (20) around the control position (22) which has a greater extent than the illumination pattern (20) previously used to determine the position of the emitter (E).
  13. Procedure according to Claim 12 , characterized in that the extent of the illumination pattern (20) used to determine the CFR value is selected such that the light intensity of the illumination light (B) at the control position (22) is above a threshold value, in particular maximized when the local minimum of the intensity distribution is arranged at the illumination positions (21), in particular wherein a distance between the illumination positions (21) and the control position (22) is approximated to a distance between the local minimum and a maximum of the intensity distribution, in particular corresponds to
  14. Procedure according to Claim 11 , characterized in that the illumination positions (21) used to determine the CFR value form an illumination pattern (20) around the control position (22) which has the same extent as the illumination pattern (20) last used to determine the position of the emitter (E).
  15. Procedure according to Claim 2 or one of the Claims 3 until 14 , insofar as it relates back to Claim 2 characterized in that the control variable is an emission frequency value or is determined on the basis of an emission frequency value.
  16. Method according to one of the preceding claims, characterized in that a vector (24) is determined during the control, the components of which are different, in particular independently determined, control variables, wherein information about the emitter (E) or the environment of the emitter (E) is collected on the basis of coordinates of the vector (24) in an n-dimensional space.
  17. Procedure according to Claim 16 , characterized in that , based on an orientation of the vector (24) relative to a hypersurface (25) of n-dimensional space, information about the emitter (E) or the environment of the emitter (E) is collected, in particular wherein the information a. indicates whether the detected light emissions originate from a single emitter (E) or from several neighboring emitters (E), and/or b. allows an assignment of the emitters (E) to one of several emitter species, and/or c. allows a determination of a state of the emitter (E), in particular a photophysical or photochemical state of the emitter (E).
  18. Procedure according to Claim 16 or 17 , insofar as it relates back to Claim 10 and 14 , characterized in that the control variables forming the components of the vector (24) include at least the CFR value and the emission frequency value.
  19. Method according to one of the preceding claims, characterized in that a sequence of localizations of an emitter (E) in the sample (2) is automatically carried out, wherein the control is carried out automatically at least once during the sequence, and wherein, based on the control, a decision is automatically made to terminate, continue and/or adapt the sequence of localizations.
  20. Device for carrying out the procedure according to one of the Claims 1 until 19 .

Description

Technical field of the invention The invention relates to a method for localizing emitters in a sample, as well as a device, a localization microscope and a computer program for carrying out the method. State of the art Localization microscopy refers to light microscopy techniques used to determine the positions of individual emitters, particularly individual light-emitting molecules such as fluorophores, separately from other neighboring emitters. For this purpose, the emitters are typically isolated within the sample; that is, conditions are created under which, at any given time, the "active," i.e., light-emitting, emitters have an average distance from each other that is at least equal to the diffraction limit, allowing the emitted light of the individual emitters to be optically separated. This can be achieved, for example, by labeling the sample with a low density of fluorescent dyes. However, for visualizing sample structures, it is advantageous to label the sample more densely with dyes, but to choose the type of dye and the chemical conditions in the sample such that the dyes alternate between an active state and a dark state (e.g., "blink"), whereby the active dyes have an average distance at any given time that is at least equal to the diffraction limit, but the distance to dyes in the dark state can certainly be smaller than the diffraction limit. From the individual, especially successively determined, localizations, i.e. the determined positions of various stationary emitters, a localization map can be created that resembles a high-resolution image of sample structures. Alternatively, for example, several locations of an emitter moving within the sample can be determined sequentially. From the temporal sequence of these locations, a trajectory of the emitter within the sample can then be determined. This process is also known as tracking. Localization microscopy includes techniques such as PALM/STORM. In this technique, the sample is labeled with blinking fluorescent emitters and illuminated in a wide field with excitation light. The fluorescent light from the sample is imaged onto a high-resolution camera, and a time series of camera images is acquired, with the time interval between images synchronized to the blinking frequency of the emitters. The diffraction distributions of the emission light from each emitter, as displayed on the camera, are then analyzed to determine the emitter positions, for example, by using a function fit. In each successive camera image, different emitters emit fluorescent light, allowing the point spread functions of the fluorescent light to be evaluated separately. Variations of the PALM/STORM technique can be found, for example, in the publications E. Betzig et al., “Imaging Intracellular Fluorescent Proteins at Nanometer Resolution” (2006), Science 313 (5793), 1642-1645, ST Hess et al., “Ultra-High Resolution Imaging by Fluorescence Photoactivation Localization Microscopy” (2006) Biophys J. 91 (11), 4258-4272 and MJ Rust et al., “Subdiffraction-limit imaging by stochastic optical reconstruction microscopy (STORM)” (2006) Nature Meth. 3, 793-796 , described. Another localization microscopy method is the so-called MINFLUX technique. Here, the sample is also labeled with isolated emitters, and excitation light is focused into the sample. The excitation light forms an intensity distribution around the focus within the sample, with a local minimum, specifically a central zero point. The intensity distribution can, for example, be a donut or a bottle beam of the excitation light. The focus of the excitation light is then positioned at illumination locations around a previously roughly estimated position of an isolated emitter using a scanning device, and light emissions from the emitter are recorded separately for the different illumination positions. From the recorded emissions and the respective positions of the minimum of the intensity distribution (known from the scan parameters), the emitter's location is then determined using a position estimation algorithm (e.g., a maximum likelihood estimator or a least mean squares estimator). The MINFLUX technique has a particular advantage over PALM/STORM in that resolutions or positional uncertainties in the single-digit nanometer range can be achieved with very few emitted photons (high photon efficiency). A first technical implementation of the MINFLUX technique is described in the publication F. Balzarotti et al. (2017) Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes, Science 355 (6325), 606-612 described, which is also the origin of the term "MINFLUX". In the publications KC Gwosch et al. (2020) MINFLUX nanoscopy delivers 3D multicolor nanometer resolution in cells, Nat. Methods, 17 (2), 217-224 and R. Schmidt et al. (2021) MINFLUX nanometer-scale 3D imaging and microsecond-range tracking on a common fluorescence microscope, Nat. Commun. 12 (1), 1478 Further developments o