Max-planck-gesellschaft Zur F&#xf6;rderung Der Wissenschaften E. V.

Klaus Gwosch

Stefan W Hell

Yvan Eilers

Francisco Balzarotti

A method of spatially measuring a plurality of nano-scale structures in a sample comprises the steps of: marking the individual structures at different locations with fluorescent markers, coupling the individual structures to individual positioning aids whose positions in the sample are known, exciting the fluorescent markers with excitation light for emission of fluorescence light, wherein an intensity distribution of the excitation light has a local minimum, arranging the local minimum at different positions in a close-up range around the position of respective positioning aid whose dimensions are not larger than the diffraction limit at the wavelength of the excitation light, registering the fluorescence light emitted out of the sample separately for the individual fluorescent markers and for the different positions of the minimum, and determining positions of the individual fluorescent markers in the sample from the intensities of the fluorescence light registered.

Method of and apparatus for spatially measuring nano-scale structures

For spatial high resolution determining a position of a singularized molecule, which is excitable with excitation light for emission of luminescence light, in n spatial dimensions in a sample, the excitation light is directed onto the sample with an intensity distribution, which has a zero point and intensity increasing regions adjoining the zero point on both sides in each of the n spatial dimensions. The zero point is arranged at not more than n&#xd7;3 different positions. The luminescence light emitted by the singularized molecule is separately registering for each of the different positions of the zero point. The position of the singularized molecule in the n spatial dimensions in the sample is deduced from intensities of the luminescence light separately registered for the not more than n&#xd7;3 different positions of the zero point.

Method of high spatial resolution determining a position of a singularized molecule which is excitable for emission of luminescence light

For spatial high resolution determining a position of a singularized molecule, which is excitable with excitation light for emission of luminescence light, in a sample, the excitation light is provided with an intensity distribution comprising an intensity increasing region with a known strictly monotonic course of an intensity of the luminescence light over a distance of the singularized molecule to a model point of the intensity distribution. The model point is arranged at different preliminary positions such that the intensity increasing region extends over a preliminary local area of the sample including the singularized molecule. From intensity values including intensities of the luminescence light separately registered for the preliminary positions of the model point, a further local area is determined which includes the singularized molecule and which is smaller than the preliminary local area. These steps are repeated using the last further local area as the next preliminary local area.

For spatial high resolution determining a position of a singularized molecule, which is excitable with excitation light for emission of luminescence light, in n spatial dimensions in a sample, a preliminary local area including the singularized molecule is determined The excitation light is directed onto the sample with an intensity distribution, which has a zero point and intensity increasing regions adjoining the zero point on both sides in each of the n spatial dimensions. At first, the zero point is arranged at preliminary positions on known sides of the preliminary local area. Then, present positions of the zero point are successively shifted into the preliminary local area in each of the n spatial dimensions depending on photons of the luminescence light which is quasi-simultaneously separately registered for the present positions of the zero point in that the zero point is repeatedly shifted between the present positions of the zero point.

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Goettingen, Germany

Klaus Gwosch