Fluorescence Lifetime Microscopy Dead Time Correction
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Solution Overview
Problem
Current fluorescence lifetime microscopy techniques face challenges in accurately measuring the lifetime of fluorescent molecules due to dead time effects in detectors and electronics, leading to pile-up errors and limitations in increasing excitation light intensity, which restricts the applicability and accuracy of time-correlated single photon counting methods.
Innovation Solution
A method that involves defining measurement intervals between consecutive excitation light pulses, summarizing detection times for recorded photons in separate data storage, and using a computing step to combine information from these stores to generate corrected histograms, accounting for dead time effects and enabling increased excitation light intensity with reduced technical effort.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If the intensity of excitation light pulses is increased to reduce measurement time, then productivity is improved, but pile-up errors increase due to dead time effects in the detector and electronics
Solution Approach 1:
The patent replaces hardware-based dead time correction (which would require complex parallelized detector and electronics systems) with a computational approach. By using a mathematical model that accounts for dead time effects in the histogram analysis, the system can process data from standard detectors while achieving accurate lifetime measurements even at high excitation intensities.
Solution Approach 2:
The patent modifies the analysis parameters by introducing dead time correction factors into the histogram evaluation model. Instead of changing the physical detector characteristics, the approach changes the mathematical parameters used to interpret the detected photon statistics, allowing accurate extraction of lifetime information despite dead time losses.
2Measurement precision
If parallelization of detector and electronics is implemented to reduce dead time effects, then measurement precision is improved, but device complexity increases significantly
Solution Approach 1:
The patent replaces hardware-based dead time correction (which would require complex parallelized detector and electronics systems) with a computational approach. By using a mathematical model that accounts for dead time effects in the histogram analysis, the system can process data from standard detectors while achieving accurate lifetime measurements even at high excitation intensities.
3Measurement precision
If a single photon counting system is used to achieve high measurement precision, then measurement precision is improved, but productivity decreases due to long recording times required
Solution Approach 1:
The patent modifies the analysis parameters by introducing dead time correction factors into the histogram evaluation model. Instead of changing the physical detector characteristics, the approach changes the mathematical parameters used to interpret the detected photon statistics, allowing accurate extraction of lifetime information despite dead time losses.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach allows for accurate determination of fluorescence lifetimes with reduced pile-up errors, enabling higher excitation light intensity and universal applicability across detector and electronic types, while providing a corrected overall histogram representation.
Implementation Method 1
a detector is used to detect fluorescence photons emitted by periodic excitation with light pulses
Implementation Method 2
fluorescence photons emitted by periodic excitation with light pulses... the time between an excitation light pulse and the subsequent fluorescence signal
Data Source
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AI summary
The invention relates to a fluorescence-lifetime microscopy method having time-correlated single-photon counting, wherein a sample (13) is periodically excited by means of excitation light pulses to emit fluorescence photons using a pulsed light source (2), wherein a measurement interval is defined between each pair of consecutive excitation light pulses, the fluorescence photons are detected by means of a detector (16) and a detector signal (17) representing the detected fluorescence photons is produced, detection times at which the fluorescence photons are detected by the detector (16) within the measurement intervals are determined on the basis of the detector signal (17), and imaging is performed on the basis of the detection times. The aim of the invention is to increase the excitation light intensity with comparatively little technical effort whilst avoiding a pile-up effect and for universal use of detector and electronics types This aim is achieved in that, in the fluorescence-lifetime microscopy method, it is determined in the respective measurement interval whether a predetermined number of fluorescence photons has been detected within the measurement interval. The detection times for all the detected photons are aggregated in a first data memory, common to a plurality of image points. The detection times of only those fluorescence photons which have been detected in the predetermined number in the respective measurement intervals are aggregated in a second data memory, common to this plurality of image points. In a computational step, the detection times aggregated in the first data memory are combined with the detection times aggregated in the second data memory. The results of this computational step are stored in a third data memory. The invention further relates to a microscope for carrying out such a fluorescence-lifetime microscopy method.