Patent ID: 7897937
Filing Date: 2011-03-01
Classification: G01N

Abstract:
1. A method of imaging a structure in a sample with three-dimensional spatial resolution, the method comprising the steps of: selecting a fluorophore from a group of photochromic fluorophores which are repeatedly temporarily transferable by means of an optical transfer signal out of a first photochromic state into a second photochromic state having fluorescence properties specific to the second photochromic state, and which display a return rate out of the second photochromic state into the first photochromic state; labelling the structure with the fluorophore; via a common objective, subjecting the sample in a spatially limited transfer-volume to the optical transfer signal transferring a portion of the fluorophore located in the transfer-volume into its second photochromic state; via the common objective, subjecting the sample in a spatially limited excitation-volume to an optical excitation signal exciting a portion of the fluorophore located in the excitation-volume and being in its second photochromic state for fluorescence, the transfer-volume and the excitation-volume having a common centre of maximum intensity of the transfer signal and of the excitation signal; wherein, at least in the direction of the optical axis of the common objective, a decrease of intensity of the transfer signal with the distance to the common centre of maximum intensity is substantially stronger than any decrease of the effective return rate of the fluorophore; detecting fluorescence light emitted by the excited fluorophore in its second photochromic state located in and about the common centre of maximum intensity of the transfer signal and the excitation signal; shifting the common centre of maximum intensity of the transfer signal and the excitation signal with regard to the sample; and repeating the preceding steps of subjecting and detecting for the shifted common centre of maximum intensity of the transfer signal and the excitation signal.