Method of determining a measurement value on the basis of single molecule events

A method of determining at least one measurement value on the basis of a plurality of single molecule events of a plurality of marker molecules of a same kind which are present in a sample, the method comprising the steps of:

• selecting the marker molecules of the same kind from a group of marker molecules, the group of marker molecules consisting of:
• marker molecules
• which have 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 in which the measurement signal necessary for determining the at least one measurement value is not obtainable from the marker molecules, and
• which are transferrable out of their non-measurable state into their
• marker molecules
• which have 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 in which the measurement signal necessary for determining the at least one measurement value is not obtainable from the marker molecules,
• which display a transfer rate out of their non-measurable state in their measurable state, and
• which are transferrable out of their measurable state into their non-measurable state by means of a physical signal,
• providing the marker molecules in the sample at such an absolute concentration that the at least one measurement value is not determinable on the basis of single molecule events of the marker molecules, if all marker molecules are in their measurable state,
• 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 on the basis of single molecule events of the marker molecules,
• imaging the measurement area on a detector detecting the measurement signal which is not spatially resolving the measurement area, and
• determining the at least one measurement value from the measurement signal detected by the detector by a method selected from a group of methods including methods of single molecule spectroscopy and methods of fluctuation analysis,
• wherein, in determining the at least one measurement value, the measurement signal from the marker molecules is evaluated independently on a location and an orientation of the marker molecules in the measurement area.