Real‐time fiber‐based fluorescence lifetime imaging with synchronous external illumination: A new path for clinical translation

Abstract

Time‐correlated single photon counting is the “gold‐standard” method for fluorescence lifetime measurements and has demonstrated potential for clinical deployment. However, the translation of the technology into clinic is hindered by the use of ultrasensitive detectors, which make the fluorescence acquisition impractical with bright lighting conditions such as in clinical settings. We address this limitation by interleaving periodic fluorescence detection with synchronous out‐of‐phase externally modulated light source, thus guaranteeing specimen illumination and a fluorescence signal free from bright background light upon temporal separation. Fluorescence lifetime maps are generated in real‐time from single‐point measurements by tracking a reference beam and using the phasor approach. We demonstrate the feasibility and practicality of this technique in a number of biological specimens, including real‐time mapping of degraded articular cartilage. This method is compatible and can be integrated with existing clinical microscopic, endoscopic and robotic modalities, thus offering a new pathway towards label‐free diagnostics and surgical guidance in a number of clinical applications.

Real-time multispectral fluorescence lifetime imaging using Single Photon Avalanche Diode arrays

“…2) reveal a well-demarcated region that is coincident with the area of collagen digestion, yielding shorter lifetime relative to non-digested areas. These results are in close agreement with previous studies of cartilage digestion 46,47,53 . The decrease in autofluorescence lifetime is more prominent in channels 1 and 2, i.e.…”

“…For two references fluorophores (POPOP in ethanol and flavin adenine dinucleotide (FAD) in purified water) we measured fluorescence lifetimes of 1.36 ± 0.04 ns and 4.02 ± 0.08 ns, respectively. These are comparable to fluorescence lifetimes of 1.32 ± 0.03 ns and 3.79 ± 0.06 ns measured in a TCSPC setup 46 .…”

“…In a previous study, we introduced a technique employing TCSPC and hybrid photon counting detectors to realize single-channel fibre-based fluorescence lifetime imaging from single-point measurements in real-time and under bright illumination of the FOV 46 . Here, we aimed to expand this technique by employing SPAD array detectors to provide multispectral resolution to the lifetime measurement and thus increase the specificity of the fluorescence output.…”

“…Augmented fluorescence lifetime and spectral imaging in real time. To generate autofluorescence lifetime and intensity maps out of single-point measurements, we used the aiming beam approach that was previously described by others 30 and expanded by our group in the context of TCSPC measurements 46 . In brief, fluorescence measurements are realized by moving the fibre probe freely across the specimen.…”

In vivo detection of murine glioblastoma through Raman and reflectance fiber-probe spectroscopies

“…However, the known literature and this study suggest that a multimodal approach-e.g., combining reflectance with laser spectroscopy-is crucial for improving sensitivity and specificity in tumor detection beyond the capability of each single technique. Further, multimodal spectroscopy could be exploited for scanning large brain areas upon implementation within real-time fiber probes 46 or imaging systems. 47 In conclusion, the in vivo results presented here represents an additional, although preliminary, encouraging step toward clinical translation and deployment of Raman and reflectance spectroscopies for a fast, label-free detection, and delineation of human brain tumors.…”