Microscope and method for wavelength-selective and high spatial resolving microscopy

摘要

A method for wavelength-selective and high spatial resolving fluorescence microscopy. In a specimen fluorescence emitters are repeatedly excited and specimen frames are produced with a microscope. The fluorescence emitters are excited to emit fluorescence radiation such that at least a sub-set is isolated in each frame and the positions of the isolated fluorescence emitters are localized with a localization precision exceeding the optical resolution and a high-resolution complete image is produced. The imaging beam path of the microscope has a spectrally selective element which, during production of the frames, generates a spectrally-dependent rotational asymmetric point-spread function, such that images of isolated fluorescence emitters have a rotational asymmetry which depends on a wavelength at which the isolated fluorescence emitters fluoresce, and the images of the isolated fluorescence emitters are analyzed with respect to rotational asymmetry and an indication of the wavelength of the isolated fluorescence emitters is derived therefrom.

主权项

1. A method for wavelength-selective and high spatial resolving fluorescence microscopy, comprising:
a) Repeatedly exciting fluorescence emitters in a specimen to emit fluorescence radiation and producing frames of the specimen with a microscope having an imaging beam path with an optical resolution, where in the fluorescence emitters are excited to emit fluorescence radiation such that at least a sub-set of the fluorescence emitters is isolated in each frame, b) Localizing the positions of the isolated fluorescence emitters in produced frames with a localization precision exceeding the optical resolution, and producing a high resolution complete image therefrom, c) wherein the imaging beam path of the microscope has a spectrally selective element during the production of the frames and further comprising generating a spectrally dependent rotational asymmetric point-spread function, with the result that images of isolated fluorescence emitters have a rotational asymmetry which depends on a wavelength at which the isolated fluorescence emitters fluoresce, and d) analyzing, in the frames the images of the isolated fluorescence emitters with respect to rotational asymmetry and deriving an indication of the wavelength of the isolated fluorescence emitters therefrom.