Ldh p c 375b

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.

Details of this setup have been published elsewhere^{16 (link),19 (link)}. The exciation is performed with two pulsed diode lasers from Picoquant, emiting at 405 nm (LDH-P-C-405B, FWHM 60 ps, Picoquant-Germany) and 375 nm (LDH-P-C-375B, FWHM 45 ps, Picoquant-Germany). The diodes are controlled by a driver (PDL-808 “Sepia”, PicoQuant GmbH, Berlin, Germany). The repetition frequency used for this study is 40 MHz.
A single-core fiber with a 200 μm diameter, is used to bring the excitation to the sample. The signal is then collected through a single-core optical fiber with a 365 μm diameter. Then the collected signal goes through a long pass filter (SR420, Semrock) to cut the signal from laser reflection. For spectral measurement, a spectrometer (QEPro 6500, Ocean Optics, 1.5 nm spectral resolution over a 365-950 nm spectral range) was used. For lifetime measurements a filter wheel (FW102C, Thorlabs, Newton, USA) with five filters (Semrock, New York, USA, 450 ± 10 nm, 520 ± 10 nm, 550 ± 30 nm, 620 ± 10 nm and 680 ± 10 nm) in front a photomultiplier link to a TCSPC acquisition card (PMA 182 and Time Harp 200, Picoquant, Germany) was used.

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).