Abstract
Autofluorescence lifetime measurements, which can provide label-free readouts in biological tissues, contrasting e.g. different types and states of tissue matrix components and different cellular metabolites, may have significant clinical potential for diagnosis and to provide surgical guidance. However, the cost of the instrumentation typically used currently presents a barrier to wider implementation. We describe a low-cost single point time-resolved autofluorescence instrument, exploiting modulated laser diodes for excitation and FPGA-based circuitry for detection, together with a custom constant fraction discriminator. Its temporal accuracy is compared against a “gold-standard” instrument incorporating commercial TCSPC circuitry by resolving the fluorescence decays of reference fluorophores presenting single and double exponential decay profiles. To illustrate the potential to read out intrinsic contrast in tissue, we present preliminary measurements of autofluorescence lifetime measurements of biological tissues ex vivo. We believe that the lower cost of this instrument could enhance the potential of autofluorescence lifetime metrology for clinical deployment and commercial development.
Highlights
Autofluorescence spectroscopy provides a non-invasive labelfree approach to characterise biological tissues ex vivo and in vivo and has shown potential to report structural and biochemical changes associated with pathological transformations
The wide-spread exploitation of autofluorescence lifetime measurements for clinical and other applications requires more compact and lower cost instrumentation compared to the current state-of-the-art
We presented a low-cost time-resolved fluorometer integrated with a fibre-optic probe that can be applied to resolve lifetime changes for the vast majority of commonly used fluorescent labels and for the most commonly studied endogenous fluorophores in biological issue
Summary
Autofluorescence spectroscopy provides a non-invasive labelfree approach to characterise biological tissues ex vivo and in vivo and has shown potential to report structural and biochemical changes associated with pathological transformations. We present the development of a low-cost time-resolved fluorometer implemented in a single point measurement instrument that utilises a modulated laser diode for excitation and a standard photon-counting photomultiplier tube (PMT) in combination with a home-made constant fraction discriminator (CFD) for time-resolved detection of the autofluorescence signal. In time-critical photon-counting applications, such as fluorescence lifetime measurements, it is essential to have a standardised approach to detect the arrival time of each photon This can be realised using constant fraction discriminators (CFDs) that indicate the centre of the electronic pulses reporting each photon. In our circuit, such discrimination is directly implemented by adjusting the voltage offset of the low-pass filtered signal This approach reduces the number of electronic components used and the complexity of the circuit, as there is no need to match the delay of the comparator that produces triggering at a constant fraction of the amplitude with a leading-edge output line. The intensity weighted mean fluorescence lifetime measured with FPGAbased detection is in reasonable agreement with the conventional TCSPC measurement and, the precision of the FPGA measurements is lower than for the conventional TCSPC system, these results show that the FPGA-based system can resolve fluorescence lifetime variations of 10s of picoseconds - even with much larger (6.25 ns) detection windows
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