Fluorescence-lifetime imaging microscopy method having time-correlated single-photon counting, which method permits higher light intensities

Abstract

The invention relates to a fluorescence-lifetime imaging microscopy method having time-correlated single-photon counting, wherein a sample (36) is periodically excited with excitation light pulses toemit fluorescence photons by means of a pulsed light source (12), wherein a measurement interval is defined between each pair of consecutive excitation light pulses, the fluorescence photons are detected by means of a detector (42) and an analog detector signal representing the detected fluorescence photons is produced, detection times at which the fluorescence photons are detected by the detector(42) within the measurement intervals are determined on the basis of the detector signal, at least one value characterizing the fluorescence decay behavior is determined on the basis of the detectiontimes of the detected fluorescence photons, and imaging is performed on the basis of the characterizing value. The analog detector signal is sampled within each measurement interval in a plurality ofsampling intervals and is converted into a series of discrete signal values associated with the individual sampling intervals. On the basis of the series of discrete signal values that belongs to theassociated measurement interval, it is determined whether more than a predefined number of fluorescence photons has been detected within the measurement interval. This measurement interval is discarded for the determination of the characterizing value if more than said predefined number of fluorescence photons has been detected.

Description

technical field [0001] The invention relates to a method of fluorescence lifetime imaging microscopy using time-correlated single photon counting according to the preamble of claim 1 and a microscope for carrying out such a method according to the preamble of claim 16 . Background technique [0002] Fluorescence lifetime imaging microscopy, FLIM for short ("fluorescence lifetime imaging microscopy") is a fluorescence microscopy imaging method based on the measurement of different lifetimes of excited states of fluorescent molecules. With the aid of the measured lifetime, for example, properties of the environment of the fluorescent molecule can be inferred, such as pH value, temperature, ion concentration, FRET transfer (FRET=" Resonance Energy Transfer", fluorescence resonance energy transfer), etc. [0003] The fluorescence lifetime can be determined directly in the time domain (“time domain lifetime measurement”) or in an alternative method in the frequency range (“freq...

AI Technical Summary

Problems solved by technology

This approach can be implemented with relatively little equipment outlay, but is limited in terms of temporal resolution and signal utilization

Time-windowed methods working with CCD or CMOS cameras have similar disadvantages

Images