Spc 730

A custom-made software developed in LabVIEW 2015 (National Instruments, Austin, TX, USA), allowed us to control the motorized stages, to set and program the features of the laser beams and the relative position between objective and sample, to start excitation and acquisition by triggering two synchronized I/O boards, and to build the images from the acquired signals. The I/O boards are a PCI-MIO (National Instruments, Austin, TX, USA) and a SPC-730 (Becker-Hickl GmbH, Berlin, Germany). The former board controls the movements of shutters, scanning mirrors and acquires the amplified and integrated signals of the PMTs; the latter board is dedicated to time-resolved single photon counting measurements. In this case, the visualization of the acquired images is accomplished using the dedicated software, SPCM 9.81 (Becker-Hickl GmbH, Berlin, Germany), that also allowed us to change the photon counting board settings. Image pixels exponential fits, de-convolution and fluorescence decay analyses are performed using the software SPC-Image 2.8 (Becker-Hickl GmbH, Berlin, Germany). Raman and fluorescence spectrum acquisitions were performed using the software Andor Solis (Andor, Belfast, UK). Images were handled with ImageJ and graphs and histograms were prepared in Microcal Origin Pro 8.1 (OriginLab Corporation, Northampton, MA, USA).

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).