Method for High-Resolution 3D-Localization Microscopy

A method for high-resolution 3D-localization microscopy of a sample comprising fluorescence emitters, in which the fluorescence emitters in the sample are excited to emit fluorescent radiation and the sample is displayed with spatial resolution in wide-field microscopy, said excitation being caused such that in reference to the spatial resolution at least some fluorescence emitters are isolated, and in a localization analysis a three-dimensional localization is determined, which includes in the depth direction of the display a z-coordinate and a x-coordinate as well as a y-coordinate orthogonal in reference thereto for each isolated fluorescence emitter, showing a precision exceeding the spatial resolution, comprising

• determining localization imprecision, which states the imprecision of the localization, namely at least regarding its z-coordinate as a function of the z-coordinate and a number of photons collected during imaging in the wide-field microscopy, and
• determining localization imprecision for each localized fluorescence emitter by accessing the table of localization imprecision for the localization determined during the localization analysis, with the localization imprecision being determined by way of simulation comprising the following steps:
• a) simulating an image for an ideal, dot-shaped fluorescence emitter for the predetermined number of photons emitted by the fluorescence emitters for an image, for an average number of photons,
• b) modifying said simulated image with a pixel confusion function to form an expanded image, with the pixel confusion function dependent on a predetermined localization of the fluorescence emitter,
• c) modifying the expanded image in consideration of statistic noise influences to form a noisy, expanded image, with the statistic noise influences being dependent on the number of photons,
• d) subjecting the expanded image to a localization analysis, in order to obtain a simulated localization from the noisy, expanded image, and
• e) repeating steps b) through d) several times for various values of the statistic noise influences, a spread of the several simulated localizations obtained here, or a spread of differences between these several simulated localizations and a predetermined localization, is determined, and from said spread the imprecision of the predetermined localization is determined.

A high-resolution microscope for 3D-localization microscopy of a sample having fluorescence emitters, comprising

• an excitation device arranged to excite the fluorescence emitters in the sample to emit fluorescent radiation and an imaging device to display the sample with a spatial resolution,
• said excitation device being arranged such that at least some of the fluorescence emitters in the display are isolated with respect to the spatial resolution, and
• a localization analysis device arranged to determine a three-dimensional localization, which comprises in the depth direction of the illustration a z-coordinate as well as a x-coordinate and a y-coordinate orthogonal in reference thereto for each isolated fluorescence emitter with a precision exceeding the spatial resolution,
• said localization analysis device having a table of localization imprecision stating the imprecision of localization, at least with regards to its z-coordinate as a function of the z-coordinate and a number of photons collected by the wide-field camera,
• said localization analysis device arranged to determine for each localized fluorescence emitter, by accessing the table of localization imprecision, a localization imprecision for the previously determined localization.