Abstract: The invention relates to a method for producing linear DNA molecules with at least two or more repeating units of a target DNA sequence for use as DNA substrate in single-molecule assays, wherein a target DNA is subjected to a ligation reaction and the ligation reaction is stopped when the reaction reaches the end of the linear phase to block circularization of the DNA molecules. Compositions comprising such linear DNA molecules and use of such compositions in single-molecule assays are also provided.

Abstract: A method of detecting a biomolecular process is provided. The method comprises: providing a DNA strand (11) comprising one or more luminophores (3) attached to the strand (11) at one or more predetermined positions along the strand; attaching handles (7A, 7B) to the DNA strand; and trapping and/or manipulating the strand (11) by manipulating the handles (7A, 7B) in a trapping setup. The method also comprises determining a position of at least one of the one or more luminophores (3) with respect to at least part of the trapping setup and determining on the basis of the one or more luminophores (3) one or more of: a focus position of at least part of an optical detector and/or an illumination system; a focus position of at least part of an optical manipulator; a position of at least part of the strand (11); a position of a portion of the strand (11) relative to one or more other portions of the strand (11); a reaction or binding position on the strand (11) of a reagent interacting with the strand (11).

Abstract: A method of excitation microscopy, in particular STimulated Emission Depletion (STED) microscopy, ins provided which comprises: providing a sample; trapping an object in the sample at a trapping position, in particular by applying a position dependent trapping force to the object; positioning, in particular focusing, a depletion beam at an interaction position in the sample for illumination of a portion of the sample associated with the trapped object.

Abstract: A method of excitation microscopy, in particular STimulated Emission Depletion (STED) microscopy,ins provided which comprises: providing a sample; trapping an object in the sample at a trapping position, in particular by applying a position dependent trapping force to the object; positioning, in particular focusing, a depletion beam at an interaction position in the sample for illumination of a portion of the sample associated with the trapped object.