Patent ID: 7202953

Claim:
Method for optically detecting at least one entity chosen from the group consisting of molecules, molecule complexes, polymers, polymeric particles, particles built up from inorganic materials, vesicular structures, cells, bacteria and virus, comprising: arranging the at least one entity on and/or in a substrate, said substrate having a refraction-index which is different from the one of an at least one component adjacent to the substrate; scanning the at least one entity with a measuring volume using at least one device being confocal or configured for multi-photon-excitation, said device comprising a first radiation source and at least one first objective, thereby receiving measuring values of optical parameters which are processed by means of signal processing for characterization of the at least one entity; the at least one entity substantially maintains its position with respect to the substrate for the duration of receiving the measuring values, generating, before and/or during the scanning step, an auxiliary focus by means of at least one second radiation source and a second objective, said auxiliary focus is at least partly arranged on an interface between substrate and adjacent component or on another interface having a defined spacial relation to said entity; collimating the radiation generated by the first radiation source by a first optic and collimating the radiation generated by the second radiation source by a second optic being different from the first optic; detecting a retroreflection from the auxiliary focus by a detector having a confocal arranged diaphragm or by a plurality of detectors having diaphragms being arranged in front of and/or behind an image plane, and along the optical axis of the objective generating the auxiliary focus, said retroreflection is used for measuring position of the interface and, thus, for indirectly positioning the measuring volume; and adjusting position of the auxiliary focus relative to the measuring volume in a defined manner.