Murine and Porcine Autofluorescence Measurement

AFL measurements from murine and porcine samples were realised using a bench-top single-point multidimensional fluorometer as previously described^{15 (link),20 (link),21 (link)}. This instrument provides spectral and time-resolved information of the fluorescence signal using an optical fibre probe extension, which facilitates measurements from tissue samples. Excitation light at 5 MHz was provided by a 375 nm laser diode (LDH-P-C-375B, PicoQuant GmbH, Germany) generating ~70 ps optical pulses. Excitation light was delivered to the sample via a custom-built fibre optic probe (NA = 0.22, FiberTech Optica, Canada) incorporating three multimode excitation fibres and sixteen multimode collection fibres. At the proximal end, fluorescence light emanating from the sample was focused onto the input slit of a motorised monochromator (CM110, CVI Inc., USA) before being directed to a cooled photomultiplier tube (PMT, PMC-100-1, Becker & Hickl GmbH, Germany) to provide single channel, spectrally resolved detection with time-resolution implemented using time-correlated single photon counting (TCSPC, SPC-730, Becker & Hickl GmbH, Germany). Autofluorescence from the specimens excited at 375 nm was measured at the emission peak wavelength, i.e. 460 nm (data not shown).