| abstract |
The invention relates to a method and an arrangementnfor the simultaneous generation of time-resolved fluorescence imagesn(Fluorescence lifetime imaging) and time-resolvednEmission spectra based on time and location correlatednSingle photon counting (ZOKEPZ) to determinenParameters for samples such as living cells, in multi-well,nin vitro fluorescence assays, in DNA chips, in which a pulsed,nhigh frequency, polarized laser beam on onenFluorescence sample is conducted and that of the fluorescence samplenemitted fluorescent light on a beam splittern(Intensity, color or polarization divider) steered andnthere is split into two beam branches.n n n The object of the invention to develop a generic method and an arrangement with which the described disadvantages of the prior art are avoided and with which information, for example, about macromolecular interactions in living cells while preserving undisturbed life by minimally invasive observation using ultra-low excitation energies of ≤ 1mW / cm2 and ultra-low dye concentrations (1 sample molecule EB in 10,000 DNA base pairs), while avoiding the artifacts caused by autofluorescence, is solved by a method which is characterized in that one beam branch (T1) through a dispersion element (11) and a neutral (12) and polarization filter (20) onto a position and time-correlated single photon count detector (13) (OZKEPZ detector) and the other beam branch (T2 ) through a neutral (14) as well as color (22) and polarization filter (21) to a second OZ KEPZ detector (15) is conducted, the detectors (13, 15) simultaneously determining the position / wavelength / polarization and time coordinates and the absolute arrival time of the individual photons (multi-parameter acquisition system), which are determined in a control - Electronics (19) and PC can be saved and evaluated. |