Fluorescence lifetime imaging microscopy method using time-correlated single-photon counting allowing higher light intensities

Abstract

A fluorescence lifetime imaging microscopy method using time-correlated single-photon counting, by means of a pulsed light source (12) to periodically excite a sample (36) with excitation light pulses to emit fluorescent photons, in every two consecutive excitations A measurement interval is defined between the light pulses; the fluorescent photons are detected by means of a detector (42) and an analog detector signal representing the detected fluorescent photons is generated; the detection time is determined based on the detector signal, and the fluorescent photons are within the corresponding measurement interval on the detection time Detected by a detector (42); based on the detection time of the detected fluorescent photons, at least one parameter characterizing the decay characteristic of the fluorescence is determined, and imaging is performed by means of the characterizing parameter. The analog detector signal is sampled in a plurality of sampling intervals within the corresponding measurement interval and converted into a sequence of discrete signal values ​​assigned to the individual sampling intervals. With the aid of the sequence of discrete signal values ​​belonging to the respective measurement interval, it is determined whether more than a predetermined number of fluorescent photons are detected within the measurement interval, and if so, the measurement interval is discarded for determining the characteristic variable.

Description

Fluorescence lifetime imaging using time-correlated single-photon counting allowing higher light intensity Microscopy technical field The present invention relates to a fluorescence lifetime imaging microscopy method utilizing time-correlated single photon counting and A microscope for carrying out such a method. Background technique Fluorescence lifetime imaging microscopy, referred to as FLIM ("fluorescence lifetime imaging microscopy") is a fluorescence microscopy imaging method based on the excitation of fluorescent molecules Measurement of different lifetimes. Using the measured lifetime, properties of the environment of the fluorescent molecule, such as pH, temperature, for example, can be deduced. degree, ion concentration, FRET transfer (fluorescence resonance energy transfer), etc. [0003] Fluorescence lifetimes can be determined directly in time scale ("time domain lifetime measurements") or in an alternative method is determined in the frequency ra...

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

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