Real-time multispectral fluorescence lifetime imaging using Single Photon Avalanche Diode arrays
Authors: Lagarto JL., Villa F., Tisa S., Zappa F., Shcheslavskiy V., Pavone FS., Cicchi R.
Autors Affiliation: 1National Institute of Optics National Research Council (INO-CNR), Largo Enrico Fermi 6, 50125, Florence, Italy.
2European Laboratory for Non-linear Spectroscopy (LENS), Via Nello Carrara 1, 50019, Sesto Fiorentino, Italy.
3Dipartimento di Elettronica, Informazione e Bioingegneria (DEIB), Politecnico di Milano, 20133, Milan, Italy. 4Micro
Photon Device SRL, Via Waltraud Gebert Deeg 3g, I-39100, Bolzano, Italy. 5Becker & Hickl GmbH, Nunsdorfer
Ring 7-9, 12277, Berlin, Germany. 6Department of Physics, University of Florence, Via G. Sansone 1, 50019, Sesto
Fiorentino, Italy. 7Privolzhskiy Medical Research University, 603005, Nizhny Novgorod, Russia.
Abstract: Autofluorescence spectroscopy has emerged in recent years as a powerful tool to report label-free
contrast between normal and diseased tissues, both in vivo and ex vivo. We report the development
of an instrument employing Single Photon Avalanche Diode (SPAD) arrays to realize real-time
multispectral autofluorescence lifetime imaging at a macroscopic scale using handheld single-point
fibre optic probes, under bright background conditions. At the detection end, the fluorescence signal is
passed through a transmission grating and both spectral and temporal information are encoded in the
SPAD array. This configuration allows interrogation in the spectral range of interest in real time. Spatial
information is provided by an external camera together with a guiding beam that provides a visual
reference that is tracked in real-time. Through fast image processing and data analysis, fluorescence
lifetime maps are augmented on white light images to provide feedback of the measurements in realtime.
We validate and demonstrate the practicality of this technique in the reference fluorophores
and in articular cartilage samples mimicking the degradation that occurs in osteoarthritis. Our results
demonstrate that SPADs together with fibre probes can offer means to report autofluorescence spectral
and lifetime contrast in real-time and thus are suitable candidates for in situ tissue diagnostics.
Journal/Review: SCIENTIFIC REPORTS
Volume: 10 Pages from: 8116 to: 8116
KeyWords: lifetime spectroscopy, multispectral imaging, SPAD arrayDOI: https://doi.org/10.1038/s41598-020-65218-3