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.

Keywords: FLIm; cardiovascular imaging; fluorescence lifetime imaging; image-guided surgery; intraoperative tumor delineation.

FIGURE 1

Tissue autofluorescence. A, Schematic of light-tissue interaction, where incident excitation light results in the generation of fluorescence emission. The tissue penetration depth depends on the tissue scattering (μ_s) and absorption (μ_a) properties, which are wavelength (λ_ex) dependent. The longer the wavelength, the deeper light penetrates. B, Simplified Jablonski diagram for fluorescence, where a photon (hν_ex) excites the electrons from the ground state (S₀) to an excited state (S₁). The radiative relaxation back to S₀ emits fluorescence photons (hν_em). C, Absorption (abs.) and emission (em.) spectra featuring the Stokes shift. D, Temporal intensity decay of the fluorescence emission characterized by the fluorescence lifetime (τ) following an excitation pulse

FIGURE 2

Fiber probe configurations compatible with clinical applications of FLIm. A, Bifurcated probes, where excitation and collection light is guided in independent optical fibers bundled together within a common jacket. B, Enface multi-mode fiber (MMF), where excitation and collection light is guided through the same optical fiber. C, Side-viewing MMF directs the excitation light at an arbitrary angle (typically 90°) through distal-end polishing or the addition of distal-end optics (eg, prism). This configuration allows for intraluminal imaging when combined with a rotation mechanism. D, Distal-end optics include GRIN lenses (terminated with a prism for side-viewing modalities), ball lenses and freeform reflective optics. E, Multimodal probes include pairing with ultrasound transducers for FLIm-IVUS, double-clad fibers for FLIm-OCT and fiber bundles for FLIm-Raman

FIGURE 3

Data processing pipeline for time domain FLIm using pulse sampling technique. A, Raw waveform, signal. B, Fiber background. C, Background subtracted from the signal. Each box outlines the signal in one spectral band (j). D, Truncated signal y_j(k). E, Instrument impulse response function (iIRF) h_j(k). F, Deconvolved fluorescence impulse response function (fIRF) I_j(k) from which to extract intensity and lifetime parameters

FIGURE 4

Overview of the key processing steps required for real-time visualization of FLIm data acquired in a clinical setting

FIGURE 5

Illustration of, A, various FLIm integration schemes, B, data visualization strategies and, C, validation against histopathology evaluation for applications in oral and oropharyngeal cancer (adapted from Reference [110, 127]), brain cancer (adapted from Reference [109]) and breast cancer (adapted from Reference [155])

FIGURE 6

Key milestones and studies of label-free time-resolved fluorescence spectroscopy and imaging for tissue assessment in oral, brain and breast cancer. FLIM, fluorescence lifetime imaging microscopy; FLIm, fluorescence lifetime imaging; GBM, glioblastoma multiforme; HGG, high-grade glioma; IR, intensity ratio; LT, lifetime; LGG, low-grade glioma; PAI, photoacoustic imaging; TRFS, time-resolved fluorescence spectroscopy; TCSPC, time-correlated single-photon counting; UBM, ultrasound backscatter microscopy

FIGURE 7

Intravascular FLIm enables label-free identification of biological species associated with plaque progression (representative results); adapted from Reference [113] Left panel: lesions consistent with new plaque formation are associated with an increase in 390 nm lifetime and intensity (AIT, adaptive intimal thickening; PIT, pathological intimal thickening). Central panel: the amount of superficial mFC assessed using a semi-quantitative scale using CD68 immunohistostaining (0, absent; 1, <10%; 2, 11%−25%; 3, 25%−50%; and 4, >50% of superficial 200 μm) is associated with a corresponding increase in 540 nm band lifetime. Right panel: this finding allowed for the creation of a predictor (piecewise linear interpolation of 540 nm lifetime) that can map the location and degree of mFC infiltration over the vessel lumen surface with high accuracy

FIGURE 8

Demonstration of applications of FLIm in vivo. A, Optimization of flushing procedures enable the acquisition of FLIm-IVUS data from coronary arteries in swine. Fluorescence obtained from the vessel wall demonstrates a consistent lifetime despite large variations in fluorescence intensity and the presence of residual blood; adapted from Reference [108]. In vivo imaging in rabbit aorta using a FLIm-OCT catheter demonstrates, B, increased lifetime in the location of a lipid-rich plaque created by balloon injury and, C, increased lifetime in locations with macrophage activity; adapted from Reference [114]