Pmc 100 1

AFL measurements of human samples were realised using a more compact fibre optic based time-resolved spectrofluorometer previously described elsewhere^{22 (link)}. The instrument comprises two excitation sources, although for the measurements reported below we only used a 375 nm laser diode (LDH-P-C-375B, PicoQuant GmbH, Germany) that provides ~70 ps optical pulses. The average power at the sample plane was kept below 25 μW. The laser repetition rate was adjusted to 5 MHz. Excitation light was delivered to the sample via a bifurcated optical fibre bundle (NA = 0.22, FiberTech Optica, Canada) that consisted of three multimode excitation fibres and fourteen multimode detection fibres. Fluorescence from the sample was delivered to the detection arm that consisted of a set of filters and dichroic mirrors that separate the signal in three bands to provide spectral discrimination: 400–420 nm (channel 1); 430–480 nm (channel 2); 500–550 nm (channel 3). Each channel has a photon-counting PMT (PMC-100–1, Becker & Hickl GmbH, Germany) connected to a TCSPC card (SPC-830, Becker & Hickl GmbH, Germany) via a router (HRT-41, Becker & Hickl GmbH, Germany). The TCSPC system records the temporal fluorescence intensity decay profile for each spectral channel.

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).

Spatially and temporally combined excitation beams were coupled into the mirror scanning unit (Nikon C1) of the confocal microscope (Nikon Eclipse TE2000-E) and focused onto the sample by a high-NA objective (Nikon Plan Fluor 40x/1.30 oil). Forward propagating CARS emission was collected with a high-NA lens, passed the filter block and was focused on a single–photon-counting photomultiplier tube (PMC-100-20, Becker & Hickl GmbH). Epi-propagating fluorescence was collected by the excitation objective and directed by dichroic mirror to the back-port of the microscope. After passing the filter block the fluorescence emission was focused on a single-photon-counting photomultiplier tube (PMC-100-1, Becker & Hickl GmbH).