US 12,259,329 B2
Method of disturbance correction, and laser scanning microscope having disturbance correction
Benjamin Harke, Göttingen (DE); Roman Schmidt, Göttingen (DE); and Lars Kastrup, Göttingen (DE)
Assigned to ABBERIOR INSTRUMENTS GMBH, Gottingen (DE)
Appl. No. 17/782,784
Filed by ABBERIOR INSTRUMENTS GMBH, Göttingen (DE)
PCT Filed Dec. 14, 2020, PCT No. PCT/EP2020/085893
§ 371(c)(1), (2) Date Jun. 6, 2022,
PCT Pub. No. WO2021/122407, PCT Pub. Date Jun. 24, 2021.
Claims priority of application No. 102019008989.8 (DE), filed on Dec. 21, 2019.
Prior Publication US 2023/0003651 A1, Jan. 5, 2023
Int. Cl. G01N 21/64 (2006.01); G02B 21/00 (2006.01)
CPC G01N 21/6458 (2013.01) [G01N 21/6428 (2013.01); G02B 21/0032 (2013.01); G02B 21/006 (2013.01); G02B 21/0072 (2013.01); G02B 21/0076 (2013.01); G01N 2021/6439 (2013.01)] 20 Claims
OG exemplary drawing
 
1. A method for determining locations of individual fluorescence dye molecules coupled to a structure in a sample comprising the steps of
sequentially illuminating on of an individual, spatially isolated fluorescence dye molecule by an intensity distribution of an excitation light comprising a local intensity minimum or by an intensity distribution of an excitation light and an intensity distribution of a fluorescence-preventing light comprising a local intensity minimum and substantially complementary to the intensity distribution of the excitation light at a sequence of different scan positions of the local intensity minimum for exciting the fluorescence dye molecule to emit fluorescence light;
detecting the number of fluorescence photons of the fluorescence light and assigning the number of fluorescence photons to the respective scan position at each step of the sequence;
determining the location of the fluorescence dye molecule from the set of associated numbers of fluorescence photons and scan positions with an accuracy below a dimension of a diffraction-limited volume in a spatial direction;
wherein during the illumination of the sample the magnitude of a disturbance is determined and a weighting factor is assigned to each element of the set of associated numbers of fluorescence photons and scan positions, the value of which is determined from the magnitude of the disturbance during the detection of the fluorescence photons at the respective scan position, and wherein the elements of the set of assigned numbers of fluorescence photons and scan positions are taken into account in accordance with their weighting factor when determining the location of the fluorescence dye molecule.