Rapid (FLASH-FLIM) imaging of protoporphyrin IX in a lipid mixture using a CMOS based widefield fluorescence lifetime imaging camera in real time for margin demarcation applications.

The fluorescence from protoporphyrin IX (PpIX) has been employed to characterise cellular activity and assist in the visualisation of tumour cells. Its formation can be induced by 5-aminolevulonic acid (5-ALA) which is metabolised by tumour cells to form PpIX. The PpIX is localised within the cells, rather than spreading into the vascular system. This, plus its photophysics, exhibits potential in photodynamic therapy. Hence its study and the ability to rapidly image its localisation is of importance, especially in the field of fluorescence guided surgery. This has led to investigations using tissue phantoms and widefield intensity imaging. Aggregation or the presence of photoproducts can alter PpIX emission, which has implications using widefield imaging and a broad wavelength range detection. The use of the fluorescence lifetime imaging (FLIM) is therefore advantageous as it can distinguish between the emissive species as they exhibit different fluorescence lifetimes. Here we use PpIX in a construct consisting of lipid mixture (Intralipid), employed to simulate fat content and optical scattering, in a gellan gum matrix. PpIX in intralipid in aqueous solution was injected into the gellan host to form inclusions. The samples are imaged using commercial widefield TCSPC camera based on a sensor chip with 192 × 128 pixels. Each pixel contains both detection and photon timing enabling the Fluorescence Lifetime Acquisition by Simultaneous Histogramming (FLASH). This 'FLASH-FLIM' approach enables widefield fluorescence lifetime images, displayed in real time to be acquired, which has potential for use in visualising tumour boundaries.

Effect of pH on aqueous phenylalanine studied using a 265-nm pulsed light-emitting diode.

Recently, we described the characteristics and application of a 265-nm AlGaN light-emitting diode (LED) operated at 1-MHz repetition rate, 1.2-ns pulse duration, 1.32-microW average power, 2.3-mW peak power, and approximately 12-nm bandwidth. The LED enables the fluorescence decay of weakly emitting phenylalanine to be measured routinely in the condensed phase, even in dilute solution. For a pH range of 1-11, we find evidence for a biexponential rather than a monoexponential decay, whereas at pH 13, only a monoexponential decay is present. These results provide direct evidence for the dominance of two phenylalanine rotamers in solution with a photophysics closer to the other two fluorescent amino acids, tyrosine and tryptophan, than has previously been reported. Although phenylalanine fluorescence is difficult to detect in most proteins because of its low quantum yield and resonance energy transfer from phenylalanine to tyrosine and tryptophan, the convenience of the 265-nm LED may well take protein photophysics in new directions, for example, by making use of this resonance energy transfer or by observing phenylalanine fluorescence directly in specific proteins where resonance energy transfer is inefficient.

Pre-denaturing transitions in human serum albumin probed using time-resolved phosphorescence.

The investigation of protein dynamics has long been of interest, since protein interactions and functions can be determined by their structure and changes in conformation. Although fluorescence, occurring on the nanosecond timescale, from intrinsic fluorescent amino acids has been extensively used, in order to fully access conformational changes longer timescales are required. Phosphorescence enables processes on the microsecond to second timescale to be accessed. However, at room temperature this emission can be weak and non trivial to measure. It requires the removal of oxygen - a common triplet state quencher and appropriate instrumentation. In this work we make use of a chemical deoxygenator to study room temperature phosphorescence from tryptophan in human serum albumin excited using a pulsed UV light emitting diode. This is extended to monitor the phosphorescence emission upon increasing temperature, allowing pre-denaturing transitions to be observed. Time-resolved data are analysed, both as the sum of exponential decays and using a distribution analysis based on non extensive decay kinetics. These results are compared to a fluorescence study and both the average lifetime and contribution of the different emitting components were found to give more dramatic changes on the phosphorescence timescale.