Artificial Neural Network (ANN)-Based Determination of Fractional Contributions from Mixed Fluorophores using Fluorescence Lifetime Measurements

<jats:title>Abstract</jats:title><jats:p>Here we present an artificial neural network (ANN)-approach to determine the fractional contributions <jats:italic>P</jats:italic><jats:sub>i</jats:sub> from fluorophores to a multi-exponential fluorescence decay in time-resolved lifetime measurements. Conventionally, <jats:italic>P</jats:italic><jats:sub>i</jats:sub> are determined by extracting two parameters (amplitude and lifetime) for each underlying mono-exponential decay using non-linear fitting. However, in this case parameter estimation is highly sensitive to initial guesses and weighting. In contrast, the ANN-based approach robustly gives the <jats:italic>P</jats:italic><jats:sub>i</jats:sub> without knowledge of amplitudes and lifetimes. By experimental measurements and Monte-Carlo simulations, we comprehensively show that accuracy and precision of <jats:italic>P</jats:italic><jats:sub>i</jats:sub> determination with ANNs and hence the number of distinguishable fluorophores depend on the fluorescence lifetimes’ differences. For mixtures of up to five fluorophores, we determined the minimum uniform spacing Δ<jats:italic>τ</jats:italic><jats:sub>min</jats:sub> between lifetimes to obtain fractional contributions with a standard deviation of 5%. In example, five lifetimes can be distinguished with a respective minimum uniform spacing of approx. 10 ns even when the fluorophores’ emission spectra are overlapping. This study underlines the enormous potential of ANN-based analysis for multi-fluorophore applications in fluorescence lifetime measurements.</jats:p&gt

Advantages and Limitations of Fluorescence Lifetime Measurements Using Single-Photon Avalanche Diode (SPAD) Array Detector: A Comprehensive Theoretical and Experimental Study

Fast fluorescence lifetime (FL) determination is a major factor for studying dynamic processes. To achieve a required precision and accuracy a certain number of photon counts must be detected. FL methods based on single-photon counting have strongly limited count rates because of the detector’s pile-up issue and are suffering from long measurement times in the order of tens of seconds. Here, we present an experimental and Monte Carlo simulation-based study of how this limitation can be overcome using array detectors based on single-photon avalanche diodes (SPADs). We investigated the maximum count rate per pixel to determine FL with a certain precision and accuracy before pile-up occurs. Based on that, we derived an analytical expression to calculate the total measurement time which is proportional to the FL and inversely proportional to the number of pixels. However, a higher number of pixels drastically increases data rate. This can be counteracted by lowering the time resolution. We found that even with a time resolution of four times the FL, an accuracy of 10% can be achieved. Taken all together, FLs between 10 ns and 3 ns can be determined with a 300-pixel SPAD array detector with a measurement time and data rate less than 1 µs and 700 Mbit/s, respectively. This shows the enormous potential of SPAD array detector for high-speed applications requiring continuous data read out.</jats:p

Advantages and Limitations of Fluorescence Lifetime Measurements Using Single-Photon Avalanche Diode (SPAD) Array Detector: A Comprehensive Theoretical and Experimental Study

Fast fluorescence lifetime (FL) determination is a major factor for studying dynamic processes. To achieve a required precision and accuracy a certain number of photon counts must be detected. FL methods based on single-photon counting have strongly limited count rates because of the detector’s pile-up issue and are suffering from long measurement times in the order of tens of seconds. Here, we present an experimental and Monte Carlo simulation-based study of how this limitation can be overcome using array detectors based on single-photon avalanche diodes (SPADs). We investigated the maximum count rate per pixel to determine FL with a certain precision and accuracy before pile-up occurs. Based on that, we derived an analytical expression to calculate the total measurement time which is proportional to the FL and inversely proportional to the number of pixels. However, a higher number of pixels drastically increases data rate. This can be counteracted by lowering the time resolution. We found that even with a time resolution of four times the FL, an accuracy of 10% can be achieved. Taken all together, FLs between 10 ns and 3 ns can be determined with a 300-pixel SPAD array detector with a measurement time and data rate less than 1 µs and 700 Mbit/s, respectively. This shows the enormous potential of SPAD array detector for high-speed applications requiring continuous data read out

Single-photon avalanche diode (SPAD) array detector for high-throughput fluorescence lifetime flow cytometry

Time-domain measurement of the fluorescence lifetime in flow cytometry offers a complementary method to traditional flow cytometry, especially for the analysis of cells and biomolecules. Short measurement times due to high throughputs (cells/s) make a suited detector mandatory for a sufficient detection of the fluorescence signal and therefore the application of this method. We summarize the requirements for a suited detector for time resolved flow cytometry (TRFC) and present a new developed SPAD array detector with 100 pixels and 340 ns time between measurement windows, allowing a high-throughput of up to 60,000 cells/s. The functionality of the detector is shown with the measurement of a 22.2 ns laser pulse, making it applicable for an improved TRFC

Hermeticity of SI1-XGEX Diaphragms for the Fabrication of a Capacitive Post-Cmos Pressure Sensor

In this work, the hermeticity of diaphragm structures is investigated and optimized. The diaphragms are developed for the monolithic post-CMOS integration of capacitive pressure sensors. Si1-XGeX is used as diaphragm material and was deposited at temperatures below 400 °C. The hermeticity of the diaphragms was evaluated at a He pressure of 1800 hPa and in a temperature range from 50 °C to about 100 °C. The diffusion coefficients were determined by measuring the changes of diaphragm deflections due to He-diffusion inside the cavity. In the CVD process of Si1-XGeX cover layer on a polycrystalline p+Si1-XGeX diaphragm for closing the etch access holes, a variation of the SiH4 and GeH4 gas flows at a substrate temperature of about 380 °C was investigated regarding the selectivity of the layer growth on different surfaces (p+Si1-XGeX, Si, and SiO2). The selectivity of the layer growth against Si and SiO2 increases with the GeH4 ratio in the process gas flow. With a pure GeH4 gas flow, an optimisation of the parameters selectivity, He-diffusion and intrinsic stress of the Si1-XGeX cover layer was found