Abstract: The invention relates to a method, a microscope (1) and a computer program for localizing and/or imaging light-emitting molecules (M) in a sample(S) contained in a sample reservoir (6) comprising illuminating the sample(S) by a light source (2) comprised in or connected to a microscope (100), detecting light emitted by the light-emitting molecules (M) in the sample(S) by a detector (3) comprised in or connected to the microscope (100), wherein the light-emitting molecules (M) comprise a bright state (B) and a dark state (D), determining, based on the detected light (F), a current value of one or more parameters, wherein at least one or the parameters co-depends on the photo-physical or photo-chemical properties of the light-emitting molecules (M) other than their average photon emission rate, and adjusting a composition of a fluid in the sample reservoir (6) to optimize the one or more parameters during the localization or imaging of the light-emitting molecules.

Abstract: The invention relates to a light microscopy method, comprising illuminating a sample (S) comprising fluorescent dye molecules (F) by an excitation light beam (14), illuminating the sample (S) by a first impeding light beam (15), generating an intensity distribution (D) of the impeding light (I) with at least one local minimum (M), providing additional impeding light (I) in an area (A) overlapping with the local minimum (M), obtaining a first fluorescence signal from the area (A) in the absence of the additional impeding light (I), and obtaining a second fluorescence signal from the area (A) in the presence of the additional impeding light (I), and correcting the first fluorescence signal by the second fluorescence signal, wherein at each point of the intensity distribution (D) at which the impeding light (I) has a light intensity (II) greater than a saturation intensity of the impeding light (I), the light intensity (II) of the impeding light (I) differs by 20% or less between the intensity distribution (D) i

Abstract: The present disclosure relates to methods and devices (1) for detecting single photons from a sample comprising at least one emitter, wherein light pulses are generated by a light source (10) to trigger the at least one emitter to emit photons, and wherein the emitted photons are detected by an acquisition system (20) comprising a detector (21), wherein a repetition rate of the light pulses or a dead time of the acquisition system is adjusted, or wherein an input comprising a desired value of the repetition rate or the dead time is received via a user interface (32), wherein a photon confidence level is determined based on the received input, and wherein an output representing said photon confidence level or indicating whether the photon confidence level is outside of a pre-determined range or a record of said photon confidence level is generated.

Abstract: The invention relates to a method for determining positions of mutually spaced molecules (M) in a sample (20), having the steps of generating (101) a plurality of light distributions, each light distribution having a local intensity minimum (110, 310) and adjacent regions (120, 320) of increasing intensity, comprising an excitation light distribution (100) and a deactivation light distribution (300); illuminating (102) the sample (20) with the excitation light distribution (100) and the deactivation light distribution (300); detecting (103) photons emitted by the molecule (M) for different positions of the excitation light distribution (100); and deriving (104) the position of the molecule (M) on the basis of the photons detected for the different positions of the excitation light distribution (100), wherein the local minimum (110) of the excitation light distribution (100) is arranged at a plurality of scanning positions (201) one after the other within a scanning region (200), and the light intensity of the

Abstract: In high spatial resolution imaging, a structure in a specimen is marked with a substance which, in a first electronic state, is excited by light of one wavelength to emit fluorescent light, which is also converted from its first into a second electronic state by that light, and which returns from its second into its first electronic state. The specimen is imaged onto a sensor at a spatial resolution not resolving an average spacing between neighboring molecules of the substance, and exposed to the light at such an intensity that the molecules in the first state are alternately excited to emit fluorescent light and converted into their second state, and that at least 10% of the molecules presently in their first state lie at a distance from their closest neighboring molecules in their first state which is greater than the spatial resolution of the imaging onto the sensor.

Abstract: For imaging of a structure, the structure is marked with a substance which can be converted by a switching signal from a first into a second state, and which provides an optical measurement signal in one of its states, only. The switching signal is applied such that at least 10% of the molecules of the substance being in the measurement signal providing state are at a distance from their closest neighbors, which is greater than the spatial resolution limit of imaging the specimen onto a sensor array, which in turn is greater than an average distance between the molecules of the substance. From an intensity distribution of the measurement signal recorded with the sensor array, the position is only determined for those molecules of the substance which are at a distance from their closest neighboring molecules in the measurement signal providing state, which is greater than the spatial resolution limit.

Abstract: For imaging of a structure, the structure is marked with a substance which can be converted by a switching signal from a first into a second state, and which provides an optical measurement signal in one of its states, only. The switching signal is applied such that at least 10% of the molecules of the substance being in the measurement signal providing state are at a distance from their closest neighbors, which is greater than the spatial resolution limit of imaging the specimen onto a sensor array, which in turn is greater than an average distance between the molecules of the substance. From an intensity distribution of the measurement signal recorded with the sensor array, the position is only determined for those molecules of the substance which are at a distance from their closest neighboring molecules in the measurement signal providing state, which is greater than the spatial resolution limit.

Abstract: In high spatial resolution imaging, a structure in a specimen is marked with a substance which, in a first electronic state, is excited by light of one wavelength to emit fluorescent light, which is also converted from its first into a second electronic state by that light, and which returns from its second into its first electronic state. The specimen is imaged onto a sensor at a spatial resolution not resolving an average spacing between neighboring molecules of the substance, and exposed to the light at such an intensity that the molecules in the first state are alternately excited to emit fluorescent light and converted into their second state, and that at least 10% of the molecules presently in their first state lie at a distance from their closest neighboring molecules in their first state which is greater than the spatial resolution of the imaging onto the sensor.

Abstract: For the high spatial resolution imaging of a structure of interest in a specimen, a substance is selected from a group of substances which have a fluorescent first state and a nonfluorescent second state; which can be converted fractionally from their first state into their second state by light which excites them into fluorescence, and which return from their second state into their first state; the specimen's structure of interest is imaged onto a sensor array, a spatial resolution limit of the imaging being greater (i.e.

Abstract: A method of determining a measurement value on the basis of a plurality of single molecule events of marker molecules in a sample comprises the steps of selecting the marker molecules from a group of marker molecules which are transferable between a measurable state in which a measurement signal necessary for determining the at least one measurement value is obtainable from the marker molecules and a non-measurable state, of providing the marker molecules in the sample at such an absolute concentration that the at least one measurement value is not determinable, if all marker molecules are in their measurable state, and adjusting a measurement concentration of the marker molecules in the measurable state by means of applying the physical signal to the sample at such an intensity that the at least one measurement value is determinable within a defined measurement area of the sample.