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US-20260126385-A1 - METHODS AND COMPOSITIONS FOR ENHANCED AND SELECTIVE PHOTOBLEACHING

US20260126385A1US 20260126385 A1US20260126385 A1US 20260126385A1US-20260126385-A1

Abstract

Disclosed herein are compositions, systems, kits, and methods for photobleaching, and selective photobleaching, a biological sample.

Inventors

  • Arne Christians
  • Thore Boettke
  • Jannik Boog
  • Oliver Braubach

Assignees

  • Bruker Nano, Inc.

Dates

Publication Date
20260507
Application Date
20251218

Claims (20)

  1. 1 . A method for detection of one or more targets in a biological sample, the method comprising: a. contacting the biological sample with a detectable label and linking the detectable label to one or more targets; b. detecting a signal from the detectable label; c. after detection at step (b), contacting the sample of (b) with PMS, PDS, a salt thereof, or any combination thereof, wherein PMS and PDS are represented by the following formulas: d. irradiating the sample of (c), wherein irradiating comprises exposing the sample to light.
  2. 2 . The method of claim 1 , wherein the light is a selected wavelength within the wavelength range required to detect the detectable label.
  3. 3 . The method of claim 1 , wherein the detectable label comprises a dye.
  4. 4 . The method of claim 1 , wherein step (a) comprises a target probe.
  5. 5 . The method of claim 1 , wherein the dye is linked to the target probe.
  6. 6 . The method of claim 1 , wherein step (a) further comprises a reporter probe, wherein the dye is linked to the reporter probe.
  7. 7 . The method of claim 1 , wherein the biological sample comprises a cell that expressed the dye.
  8. 8 . The method of claim 1 , wherein the target comprises RNA or DNA.
  9. 9 . The method of claim 1 , wherein the target comprises a protein.
  10. 10 . The method of claim 1 , wherein the target comprises a protein, and the target probe comprises an antibody.
  11. 11 . The method of claim 1 , wherein step (d) is carried out at a pH in the range of about 5 to about 9.
  12. 12 . The method of claim 1 , wherein step (c) comprises PMS or a salt thereof.
  13. 13 . The method of claim 1 , wherein step (c) comprises PDS or a salt thereof.
  14. 14 . The method of claim 1 , wherein the method does not comprise addition of an acid or an anti-oxidant buffer after step (b).
  15. 15 . The method of claim 1 , wherein the detectable label comprises a stain and wherein the stain comprises one or more of hematoxylin and eosin.
  16. 16 . The method of claim 1 , wherein irradiating is done with white light.
  17. 17 . A method for sequential detection of multiple targets in a biological sample, the method comprising: a. contacting a first target probe to the sample, wherein the first target probe is specific for a first target; b. hybridizing or binding the first target probe to the first target in the sample; c. detecting a first fluorescent detectable label, wherein the first fluorescent detectable label is linked to the first target probe; d. after detection at step (c), contacting the sample of (c) with PMS, PDS, a salt thereof, or any combination thereof, wherein PMS and PDS are represented by the following formulas: e. irradiating the sample of (d), wherein irradiating comprises exposing the sample to light; f. repeating steps (a)-(e), comprising an Nth target probe and an Nth fluorescent detectable label different from the first probe and first fluorescent detectable label.
  18. 18 . The method of claim 17 , wherein the fluorescent detectable label is chemically linked to the target probe.
  19. 19 . The method of claim 17 , wherein step (c) comprises a reporter probe, wherein the fluorescent detectable label is linked to the reporter probe and wherein the reporter probe is configured to hybridize or bind to the target probe.
  20. 20 . The method of claim 17 , wherein step (e) is carried out at a pH in the range of about 5 to about 9.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of International Application PCT/US2025/038528, filed Jul. 21, 2025, which claims the benefit of U.S. Application 63/674,750, filed Jul. 23, 2024. The contents of the aforementioned applications are incorporated herein by reference in their entireties. BACKGROUND Various methods may be used in biology and in medicine to observe different targets in a biological sample. For example, analysis of proteins and nucleic acids in various tissue or cell preparations may be performed using the techniques of histochemistry, immunohistochemistry (IHC), immunofluorescence (IF), and numerous nucleic acid hybridization, amplification, and visualization techniques. Analysis of proteins and nucleic acids in biological samples may also be performed using solid-state assays, for example, using the techniques of western and northern blots. Many of the current techniques may detect only a few targets at one time (such as immunohistochemistry (IHC) or fluorescence-based western blots where the number of targets detectable is limited by the fluorescence-based detection system) in a single sample. Further analysis of targets may require use of additional biological samples from the source, limiting the ability to determine relative characteristics of the targets such as the presence, absence, concentration, and/or the spatial distribution of multiple biological targets in the biological sample. Moreover, in certain instances, a limited amount of an individual sample may be available for analysis, or the individual sample may require further analysis. Thus, methods, agents, and devices capable of iteratively analyzing an individual sample are needed. SUMMARY Disclosed herein are methods and compositions useful in the detection, visualization, and/or characterization of target molecules in a biological sample. The methods and compositions include PMS, PDS, a salt thereof, or any combination thereof, wherein PMS and PDS are represented by the following formulas: In some embodiments, a method for detection of one or more targets in a biological sample is provided, the method comprising: (a) contacting a biological sample with a detectable label and linking the detectable label to the one or more targets, (b) detecting a signal from the detectable label, (c) after detection at step (b), contacting the sample of (b) with PMS, PDS, a salt thereof, or any combination thereof, wherein PMS and PDS are represented by the following formulas: (d) irradiating the sample of (c), wherein irradiating comprises exposing the sample to light, optionally, wherein the light is a selected wavelength range, wherein the selected wavelength range is within the wavelength range required to detect the detectable label; and (e) optionally, repeating steps (a)-(d). In some embodiments, a method for the sequential detection of multiple targets in a biological sample is provided, the method comprising: (a) contacting a first target probe to the sample, wherein the first target probe is specific for a first target; (b) hybridizing or binding the first target probe to the first target in the sample; (c) detecting a first fluorescent detectable label, wherein the first fluorescent detectable label is linked to the first target probe; (d) after detection at step (c), contacting the sample of (c) with PMS, PDS, a salt thereof, or any combination thereof, wherein PMS and PDS are represented by the following formulas: (e) irradiating the sample of (d), wherein irradiating comprises exposing the sample to light, optionally wherein the light is a selected wavelength range of light, wherein the selected wavelength range is within the wavelength range required to detect the first fluorescent detectable label; (f) repeating steps (a)-(e), comprising an Nth target probe and an Nth fluorescent detectable label different from the first probe and first fluorescent detectable label, optionally, wherein the selected wavelength range of light is within the wavelength range required to detect the Nth fluorescent detectable label. In some embodiments, a method for detecting a plurality of targets in a biological sample is provided, the method comprising: (a) contacting a plurality of target probes to the sample comprising N subsets of target probes, wherein each probe of the subsets of target probes is specific for different target; (b) hybridizing or binding the target probes to the plurality of targets in the sample; (c) detecting a first fluorescent detectable label, wherein the first fluorescent detectable label is linked to a first subset of the plurality of target probes; (d) after detection at step (c), contacting the sample of (c) with PMS, PDS, a salt thereof, or any combination thereof, wherein PMS and PDS are represented by the following formulas: (e) irradiating the sample of (d), wherein irradiating comprises exposing the sample to a light, optionally wherein the light is a selected wavelength r