Optical microscopy with phototransformable optical labels

摘要

Imaging a sample that includes phototransformable optical labels (“PTOLs”) with an optical system having a diffraction-limited resolution volume (DLRV), includes providing activation radiation to the PTOLs to activate a statistical subset of the PTOLs. A density of the PTOLs of the activated subset is less than an inverse of the DLRV. Excitation radiation is provided to the activated subset to excite activated PTOLs. Radiation emitted from the activated and excited PTOLs located at different focal planes of the optical system within the sample is detected with the optical system. The preceding steps are repeated one or more times, each time activating a different statistical subset of the plurality of PTOLs. Three-dimensional locations within the sample are determined, with a sub-diffraction-limited accuracy, of the activated and excited PTOLs based on the radiation emitted from the activated and excited PTOLs that is detected from the different focal planes of the optical system.

主权项

1. A method comprising:
providing activation radiation to a sample, wherein the sample includes phototransformable optical labels (“PTOLs”) distributed in the sample such that separation distances between PTOLs are less than a diffraction limited resolution of a detection objective of imaging optics used to image radiation from the PTOLs, to activate a subset of the PTOLs in the sample; providing excitation radiation to the sample to excite at least some of the activated subset of PTOLs; detecting, with the imaging optics, radiation emitted from the activated and excited PTOLs within the subset of PTOLs; controlling the activation radiation such that adjacent activated PTOLs in a focal plane of the detection objective of the imaging optics are separated by distances generally larger than the diffraction limited resolution of the detection objective; determining locations of the PTOLs based on the detected radiation; and creating a superresolution image of the sample based on the determined locations of the PTOLs.