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CN-116235093-B - Method, apparatus and computer program for locating emitters in a sample

CN116235093BCN 116235093 BCN116235093 BCN 116235093BCN-116235093-B

Abstract

The invention relates to a method for locating an emitter (F) in a sample (S), comprising irradiating the sample (S) with a stationary annular excitation beam (E), acquiring fluorescent photons, and estimating the position of the emitter (F) in the sample (S) from the acquired fluorescent photons. The invention also relates to a device (1) for locating an emitter (F) in a sample (S), comprising illumination means (10), acquisition means (20) and processing means (30), and to a computer program comprising instructions for causing the device (1) to execute a method for locating an emitter (F).

Inventors

  • R. SCHMIDT
  • W. Villemer

Assignees

  • 阿贝锐纳仪器有限公司

Dates

Publication Date
20260505
Application Date
20210806
Priority Date
20210113

Claims (17)

  1. 1. A method for locating an emitter (F) in a sample (S), comprising: irradiating the sample (S) with a stationary annular excitation beam (E), Obtaining fluorescent photons, and Estimating the position of the emitter (F) in the sample (S) from the acquired fluorescence photons, Wherein the method comprises sequentially projecting pinholes (21) into a first detection position (101), the pinholes (21) being arranged in the detection light path of the device (1) in a confocal plane between the sample (S) and the acquisition means (20), and wherein the excitation beam (E) is kept stationary using a first scanning device (16) and a second scanning device (12) cooperating, the first scanning device (16) sequentially placing the projections of the pinholes (21) at selected positions of a pinhole track, the second scanning device (12) inducing a reverse movement of the excitation light such that an excitation ring remains stationary in the sample (S), and wherein the spatial distribution of fluorescent photons emitted by the emitters (F) is acquired and the positions of the emitters (F) are estimated from the detected light intensities or photon counts and the positions of the respective detector elements.
  2. 2. The method of claim 1, wherein the fluorescent photons are acquired in a location-specific manner.
  3. 3. Method according to claim 2, characterized in that the position-specific acquisition of the fluorescent photons is performed in an Image Plane (IP) which is arranged confocal with respect to the plane of the Excitation Focus (EF) of the excitation beam (E).
  4. 4. Method according to claim 1, characterized in that the pinhole (21) is moved on a circular trajectory (T) around the center (C) or is placed sequentially on at least three of the first detection positions (101) on a circular trajectory (T).
  5. 5. Method according to claim 4, characterized in that the first detection positions (101) are evenly spaced on the circular trajectory (T).
  6. 6. Method according to claim 4, characterized in that the circular excitation beam (E) comprises a maximum to maximum distance (6), wherein the diameter (d) of the circular trajectory (T) is 50% to 120% of the maximum to maximum distance (6).
  7. 7. Method according to claim 4, characterized in that the diameter (d) of the circular trajectory (T) is 50% to 100% of the full width at half maximum of the detection point spread function of the emitted light.
  8. 8. The method according to one of claims 1 to 7, characterized in that the first scanning device (16) is a galvanometer scanner and/or the second scanning device (12) is an electro-optical scanner.
  9. 9. The method according to one of claims 1 to 7, characterized in that the steps of irradiating the sample with the stationary annular excitation beam (E) and acquiring the fluorescent photons are repeated, wherein the center of the annular excitation beam (E) is positioned at the respective Scanning Position (SP) in each repetition.
  10. 10. The method according to claim 8, characterized in that the steps of irradiating the sample with the stationary annular excitation beam (E) and acquiring the fluorescent photons are repeatedly performed, wherein the center of the annular excitation beam (E) is positioned at the respective Scanning Position (SP) in each repetition.
  11. 11. Method according to claim 10, characterized in that the Scanning Positions (SP) are arranged on a grid (G).
  12. 12. Method according to claim 11, characterized in that the mutual distance between the Scanning Positions (SP) is between 10% and 50% of the excitation wavelength of the excitation beam (E).
  13. 13. The method according to one of claims 1 to 7 or 10 to 12, characterized in that the estimated position of the emitter (F) is determined in a pre-positioning step followed by a MINFLUX positioning step, wherein the MINFLUX positioning step comprises irradiating the sample (S) with the annular excitation beam (E), positioning the annular excitation beam (E) at a plurality of second detection positions (201) of a second target coordinate pattern (200) based on the estimated positions determined in the pre-positioning step, acquiring fluorescent photons for each of the second detection positions (201), and estimating the position of the emitter (F) in the sample (S) with a higher resolution than in the pre-positioning step from the acquired fluorescent photons.
  14. 14. The method according to claim 8, characterized in that the estimated positions of the emitters (F) are determined in a pre-positioning step followed by a MINFLUX positioning step, wherein the MINFLUX positioning step comprises irradiating the sample (S) with the annular excitation beam (E), positioning the annular excitation beam (E) at a plurality of second detection positions (201) of a second target coordinate pattern (200) based on the estimated positions determined in the pre-positioning step, acquiring fluorescent photons for each of the second detection positions (201), and estimating the position of the emitters (F) in the sample (S) with a higher resolution than in the pre-positioning step from the acquired fluorescent photons.
  15. 15. The method according to claim 9, characterized in that the estimated positions of the emitters (F) are determined in a pre-positioning step followed by a MINFLUX positioning step, wherein the MINFLUX positioning step comprises irradiating the sample (S) with the annular excitation beam (E), positioning the annular excitation beam (E) at a plurality of second detection positions (201) of a second target coordinate pattern (200) based on the estimated positions determined in the pre-positioning step, acquiring fluorescent photons for each of the second detection positions (201), and estimating the position of the emitters (F) in the sample (S) with a higher resolution than in the pre-positioning step from the acquired fluorescent photons.
  16. 16. A device (1) for locating an emitter (F) in a sample (S), the device (1) comprising: -an illumination device (10), the illumination device (10) being configured to illuminate the sample (S) with a stationary annular excitation beam (E); an acquisition device (20), the acquisition device (20) being configured to acquire fluorescent photons; A processing device (30), the processing device (30) being configured to estimate the position of the emitter (F) in the sample (S) from the acquired fluorescence photons, and Projection means configured to sequentially project a pinhole (21) into a first detection position (101), the pinhole (21) being arranged in a detection light path of the device (1) in a confocal plane between the sample (S) and the acquisition means (20), wherein the device (1) comprises a first scanning device (16) and a second scanning device (12) configured to co-operate to keep the excitation beam (E) stationary, the first scanning device (16) sequentially placing the projection of the pinhole (21) on a selected position of a pinhole track, the second scanning device (12) inducing a reverse movement of excitation light such that an excitation ring remains stationary in the sample (S), and wherein a spatial distribution of fluorescent photons emitted by the emitter (F) is acquired and the position of the emitter (F) is estimated from the detected light intensity or photon count and the position of the corresponding detector element.
  17. 17. A computer program comprising instructions which, when executed by at least one processor, cause an apparatus (1) according to claim 16 to perform the following steps for locating an emitter (F) in a sample (S): irradiating the sample (S) with a stationary annular excitation beam (E), Obtaining fluorescent photons, and Estimating the position of the emitter (F) in the sample (S) from the acquired fluorescent photons.

Description

Method, apparatus and computer program for locating emitters in a sample Technical Field The present invention relates to a method, apparatus and computer program for locating emitters in a sample. In particular, the method may be applied to pre-positioning of an emitter in MINFLUX positioning, wherein a position estimate obtained by the method according to the present invention may be used as a starting point for MINFLUX positioning. Prior Art In the prior art, for example, in patent applications DE 10 2011 055 367 A1, WO 2015/052186 A1, patent publications DE 10 2013 114 860 B3 and Balzarotti F, eilers Y, gwosch KC,"Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes"（arXiv:1611.03401[physics.optics]）（2016 Of a, WESTPHAL V, stefani F, elf J, hell SW) has described localization and single molecule tracking methods based on the MINFLUX procedure. Essentially, a sample containing fluorophores is detected by the intensity distribution of excitation light that excites the fluorophores, triggering the emission of fluorescent photons. The intensity distribution has a local minimum (in particular an intensity near zero at the center of the distribution) and a maximum on both sides. Such a distribution is for example known from STED microscopy, wherein an intensity distribution (e.g. a 2D or 3D circle (donut)) of the evanescent light (reconstruction light) with a central minimum is formed to deplete the fluorescence signal from the off-centered fluorophores, increasing the effective resolution beyond the diffraction limit. However MINFLUX nanometer microscopy exploits the fact that if the fluorophore of interest is located exactly at the minimum of the excitation light distribution, a minimum fluorescence signal (ideally zero) is obtained, whereas the fluorescence signal increases as the distribution moves away from the minimum. In MINFLUX positioning, the excitation light distribution is positioned sequentially at several positions (forming a so-called "target coordinate pattern (targeted coordinate pattern)", TCP), also called "target coordinate set", STC) close to the expected position of the fluorophore, the fluorescence signal is measured at each of these positions, and the position estimate of the fluorophore is determined from the measured fluorescence signal and the corresponding position of the excitation light distribution, e.g. by a least mean square estimator. The process is typically iterated, i.e. repeated by placing the minimum of the excitation light distribution at a position around the position estimate obtained in the previous step. In particular, the minimum value is placed within a radius around the current position estimate, wherein the radius is reduced in each step, which results in an improved effective resolution in each step, converging to a value as low as 1nm depending on the sample and measurement parameters. Furthermore, MINFLUX positioning has a very high photon efficiency, since as the position of the excitation minima gets closer to the actual position of the fluorophore, less light is emitted by the fluorophore. By sequentially determining the positions of several single fluorophores in the sample using the MINFLUX protocol, a fluorescence image of the sample with a resolution in the range of 1nm can be constructed. In a related art, known as MINFLUX tracking, the trajectory of a single fluorophore is tracked over time by sequential MINFLUX localization that is optimized for speed rather than accuracy. In both methods, the excitation beam is preferentially moved to the position of the TCP relative to the sample by a fast beam deflection device, such as an electro-optic deflector, to reduce the measurement time. In order to locate or track fluorescent molecules using MINFLUX, it is necessary to identify single fluorophores in the sample and obtain initial positional information about the identified single molecules. TCP may then be placed in the vicinity of the initial position estimate and the minimum of the excitation light distribution may be sequentially moved to the position of TCP as described above. For example, the initial position of the fluorophore can be determined by wide field illumination of the sample and by low resolution fluorescence detection by a camera, e.g., in a manner similar to PALM/STORM microscopy. This requires a microscope system that can be switched rapidly between broad field illumination and confocal illumination with a circular excitation beam, which is technically demanding, especially considering that switching needs to occur before each localization of an individual fluorophore, which can slow down the localization process significantly. Furthermore, high photon counts are necessary for camera detection of fluorescence, so fluorophores must be exposed to excitation light for a long period of time or at high intensity. This either slows down the localization process or is subject to the risk of photobleach