Mesoscopic fluorescence lifetime imaging: Fundamental principles, clinical applications and future directions

Abstract

Fluorescence lifetime imaging (FLIm) is an optical spectroscopic imaging technique capable of real‐time assessments of tissue properties in clinical settings. Label‐free FLIm is sensitive to changes in tissue structure and biochemistry resulting from pathological conditions, thus providing optical contrast to identify and monitor the progression of disease. Technical and methodological advances over the last two decades have enabled the development of FLIm instrumentation for real‐time, in situ, mesoscopic imaging compatible with standard clinical workflows. Herein, we review the fundamental working principles of mesoscopic FLIm, discuss the technical characteristics of current clinical FLIm instrumentation, highlight the most commonly used analytical methods to interpret fluorescence lifetime data and discuss the recent applications of FLIm in surgical oncology and cardiovascular diagnostics. Finally, we conclude with an outlook on the future directions of clinical FLIm.

Light and Autofluorescence, Multitasking Features in Living Organisms

“…Besides fluorescence lifetime detection, more generally advances in fluorescence analysis are offered for example by using multiphoton excitation and data processing procedures, allowing to detect separately specific additional fluorophores according to their spectral position or their time decay, improving their in situ and real time topological localization. Applications vary from basic research to the facilitation of histopathological diagnosis, intraoperative imaging and surgeon guidance [285,[311][312][313][314][315][316][317][318][319][320][321].…”

Autofluorescent Biomolecules in Diptera: From Structure to Metabolism and Behavior

“…For example, the multiphoton excitation using the sequence of photons in the red or near infrared spectral interval provides the energy equivalent to the violet or blue spectral interval, with the advantages of reaching deeper layers of the structure under study and decreasing the risk of photobleaching and UV radiation damage. The continuous development of sophisticate devices improves also the frequency and time resolution of the AF signals, advancing AF conventional imaging to multispectral, hyperspectral and lifetime imaging procedures for the detection and topological localization of specific fluorophores in living cells and tissues, up to the label-free mesoscopic applications in clinical diagnosis [ 3 , 4 ]. It is also worth to recall the development of procedures based on the multicomponent analysis transforming the fluorescence lifetimes in a phasor plot, which have been demonstrated to effectively solve different molecular species within single pixels.…”

Intraoperative delineation of p16+ oropharyngeal carcinoma of unknown primary origin with fluorescence lifetime imaging: Preliminary report

“…FLIm has also demonstrated potential for disease diagnosis and surgical guidance in other surgical areas, including cardiovascular disease, 21–23 breast cancer, 24 brain cancer, 25 and intraoperative guidance during thyroid surgery 26 . While FLIm technology is still in investigational stages from a clinical standpoint, significant developments in optical hardware, classification methods, and computing power are rapidly facilitating FLIm towards clinical adoption 27 …”

“…While FLIm has not been specifically developed as a tool for identifying unknown primary tumors, the recent success of this technology for demarcating conventional primary oropharyngeal tumors 19,20,27 has motivated the present investigation of discriminatory performance in HNSCCUP patients. Specifically, we investigated whether the addition of FLIm to standard diagnostic workups could enhance the detection of primary lesions within p16+ HNSCCUP patients.…”