Multispectral Depth-Resolved Fluorescence Lifetime Spectroscopy Using SPAD Array Detectors and Fiber Probes.

João L Lagarto,Franco Zappa,Vladislav Shcheslavskiy,Simone Tisa,Riccardo Cicchi,Francesco Saverio Pavone,Federica Villa,Caterina Credi

doi:10.3390/s19122678

Abstract

Single Photon Avalanche Diode (SPAD) arrays are increasingly exploited and have demonstrated potential in biochemical and biomedical research, both for imaging and single-point spectroscopy applications. In this study, we explore the application of SPADs together with fiber-optic-based delivery and collection geometry to realize fast and simultaneous single-point time-, spectral-, and depth-resolved fluorescence measurements at 375 nm excitation light. Spectral information is encoded across the columns of the array through grating-based dispersion, while depth information is encoded across the rows thanks to a linear arrangement of probe collecting fibers. The initial characterization and validation were realized against layered fluorescent agarose-based phantoms. To verify the practicality and feasibility of this approach in biological specimens, we measured the fluorescence signature of formalin-fixed rabbit aorta samples derived from an animal model of atherosclerosis. The initial results demonstrate that this detection configuration can report fluorescence spectral and lifetime contrast originating at different depths within the specimens. We believe that our optical scheme, based on SPAD array detectors and fiber-optic probes, constitute a powerful and versatile approach for the deployment of multidimensional fluorescence spectroscopy in clinical applications where information from deeper tissue layers is important for diagnosis.

Highlights

Autofluorescence spectroscopy explores the optical properties of endogenous molecules, such as collagens, reduced nicotinamide adenine dinucleotide (NAD(P)H), or flavin adenine nucleotide (FAD), to provide label-free optical contrast owing to biochemical or structural transformations.The sensitivity of autofluorescence measurements to changes in the molecular environment permitsSensors 2019, 19, 2678; doi:10.3390/s19122678 www.mdpi.com/journal/sensorsSensors 2019, 19, 2678 a robust characterization of cells and tissues, and its potential for medical diagnosis has been intensively investigated in a number of applications [1,2,3,4,5,6,7]
We explored the potential of Single Photon Avalanche Diode (SPAD) arrays to realize parallel multidimensional fluorescence measurements
FAD and POPOP embedded in agarose demonstrated that SPAD arrays can report lifetime and spectral contrast from fluorescence signals emanating at different depths relative to the source and detector fibers

Summary

Introduction

Autofluorescence spectroscopy explores the optical properties of endogenous molecules, such as collagens, reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H), or flavin adenine nucleotide (FAD), to provide label-free optical contrast owing to biochemical or structural transformations.The sensitivity of autofluorescence measurements to changes in the molecular environment permitsSensors 2019, 19, 2678; doi:10.3390/s19122678 www.mdpi.com/journal/sensorsSensors 2019, 19, 2678 a robust characterization of cells and tissues, and its potential for medical diagnosis has been intensively investigated in a number of applications [1,2,3,4,5,6,7]. The potential of multispectral fluorescence lifetime spectroscopy and imaging for clinical applications has been investigated in a number of studies [8,9,10,11,12,13,14], yet it remains relatively unexploited when compared to steady-state measurements alone This is essentially due to the complexity and cost of the instrumentation required to realize multispectral and time-resolved measurements, which typically involves multiple single-anode detectors [13], multi-anode detectors [15], monochromators [16], or other complex optical multiplexing strategies [17], making the implementation of such devices technically challenging. An additional limitation refers to the long integration times typically required to realize acquisition with a reasonable number of photons over the entire spectral range

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