Fluorescence Lifetime Microscopy Photon Pile-up Reduction
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Solution Overview
Problem
Conventional fluorescence lifetime microscopy methods face challenges in detecting multiple photons within a measurement interval due to detector dead time, leading to a pile-up effect and reduced excitation light intensity to avoid this issue, which increases recording time and limits method applicability.
Innovation Solution
The method involves subdividing the measurement interval into smaller sampling intervals, digitizing the detector signal, and discarding intervals with more than a predetermined number of detected photons to prevent pile-up, allowing for higher excitation light intensity while maintaining accurate fluorescence decay behavior characterization.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If the excitation light intensity is increased to improve photon yield, then the recording time can be reduced, but the pile-up effect occurs due to detector dead time
Solution Approach 1:
The measurement interval is divided into multiple time bins, allowing the detection and separate evaluation of multiple photons within a single excitation cycle. This segmentation enables higher excitation intensities without pile-up effects because photons are sorted into different time bins based on their arrival times, rather than being rejected due to detector dead time.
Solution Approach 2:
The method uses periodic excitation light pulses to excite the sample, with each pulse initiating a new measurement interval. This periodic action allows for systematic collection of fluorescence photons over multiple cycles, accumulating sufficient statistical data for accurate lifetime measurement while maintaining high photon yield through optimized pulse timing and intensity.
2Measurement precision
If the excitation light intensity is reduced to avoid pile-up effect, then measurement accuracy is maintained, but the recording time increases significantly
Solution Approach 1:
By segmenting the measurement interval into time bins and accepting multiple photons per excitation cycle (sorting them into different bins), the method achieves both high photon yield and measurement accuracy. This eliminates the need to reduce excitation intensity, thereby avoiding extended recording times.
Solution Approach 2:
The method changes the parameter of excitation light intensity to optimal levels that maximize photon yield, while compensating for the resulting multiple photon detections through time binning. This parameter optimization reduces recording time while maintaining measurement accuracy through the mathematical processing of multi-photon events.
3Measurement precision
If conventional detectors are used with their inherent dead time, then single photon detection is enabled, but multiple photon detection within a measurement interval is restricted
Solution Approach 1:
The measurement interval is segmented into multiple time bins, allowing multiple photons to be detected and sorted within a single excitation cycle. This segmentation bypasses the detector dead time limitation by distributing multiple photons across different time bins, thereby increasing the effective photon detection rate while maintaining single-photon detection precision.
Solution Approach 2:
The method performs preliminary time-stamping and binning of detected photons before final analysis. By pre-sorting photons into time bins based on their arrival times, the system prepares the data in a format that enables efficient processing of high photon rates without losing single-photon detection capability.
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 increases the photon yield by a factor of 3.7 compared to conventional methods, enabling optimal light intensity use without pile-up effects, thereby reducing recording time and enhancing method applicability.
Implementation Method 1
a sample is periodically excited by means of a pulsed light source with excitation light pulses to emit fluorescence photons
Implementation Method 2
the fluorescence photons are detected by means of a detector and an analog detector signal representing the detected fluorescence photons is generated
Data Source
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AI summary
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 to emit 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 detection times 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 of sampling 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 the associated 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.