Fluorescence lifetime in cardiovascular diagnostics

Abstract

We review fluorescence lifetime techniques including time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) and fluorescence lifetime imaging microscopy (FLIM) instrumentation and associated methodologies that allow for characterization and diagnosis of atherosclerotic plaques. Emphasis is placed on the translational research potential of TR-LIFS and FLIM and on determining whether intrinsic fluorescence signals can be used to provide useful contrast for the diagnosis of high-risk atherosclerotic plaque. Our results demonstrate that these techniques allow for the discrimination of important biochemical features involved in atherosclerotic plaque instability and rupture and show their potential for future intravascular applications.

Figure 1

Schematic of the simultaneous time- and wavelength resolved fluorescence spectroscopy (STWRFS) system. By combining multiple bandpass and dichroic filters (405∕40, 460∕50, and 550∕50) with different lengths of optical fiber (1, 10, and 19 m) acting as optical delay, this system enables the near real-time acquisition and characterization of time-resolved fluorescence spectra using a single detector and excitation input. The recording of multiple fluorescence response pulses at selected wavelengths can be completed in hundreds of nanoseconds, which provides the capability of a real-time characterization of tissues. Configuration adapted for single-fiber excitation-collection geometry. The initial system was reported in Ref. .

Figure 2

Schematic of the FLIM experimental setup to transmit the 4-mm field of view at 4-mm working distance through the 10,000 coherent fiber bundle (0.5-mm outer bundle diameter) attached to the graded index (GRIN) objective lens and onto the gated camera. Adapted from Ref. .

Figure 3

(a) Picture, (b) schematic, and (c) cross section of the three components: 400-μm side-viewing optic fiber (OF); 3 Fr IVUS catheter; and 356-mm steering wire. These three components are integrated in a 5.4-Fr multimodal catheter (MMC). (b) and (c) show deployment of lateral (steering) movement with the steering wire (SW). The normally compact multimodal catheter components are separated in the photo for clearer viewing. The SW in the deployed state (b) is near the IVUS sheath and is mounted so that deployment by pushing on the wire will move the multimodal catheter close to the target. Adapted from Ref. .

Figure 4

Left panels: Spectroscopic parameters as a function of emission wavelength derived from the time-resolved spectra measured in ex vivo samples. Right panels: Statistical group comparisons (mean±S.E.) for specific wavelengths. Spectroscopic parameters: normalized intensity (a), average lifetime (b), LEC-0 (c), and LEC-2 (d). Adapted from Ref. .

Figure 5

FLIM images of normal artery at (a) 377∕50 nm (center wavelength∕bandwidth) and at (b) 460∕60 nm. Note the uniformly ∼2-ns average lifetimes, which is typical of elastin fluorescence. Images of atherosclerotic artery at (c) 377∕50 nm and at (d) 460∕60 nm. Note the longer lifetimes in (c) typical of collagen fluorescence and the shorter lifetimes in (d) typical of lipid fluorescence, indicating that this plaque has both collagen-rich (ROI 2) and lipid-rich (ROI 1) components. Histograms correspond to the images to their left.

Figure 6

TR-LIFS and IVUS using the multimodal catheter (a) ex vivo and (b) in vivo. (a) Top panel: Comparison fluorescence emission spectra from an ex vivo pig femoral artery for moving whole blood (N=7) conditions versus normalized spectral emission in saline (N=5). Bottom panel: The corresponding fluorescence average lifetimes along the emission spectrum. (b) Top panel: Comparison fluorescence intensity spectrum from in vivo pig femoral artery when the catheter is fully deployed and in contact with the arterial wall (N=4) versus fluorescence intensity spectrum when the catheter is not fully deployed and not in contact with the arterial wall. Bottom panel: The corresponding fluorescence lifetimes along the emission spectrum. (c) IVUS image and (d) illustration demonstrating the application of the multimodal catheter in vivo in pig femoral artery. The depicted condition corresponds to catheter in contact with the arterial wall. Adapted from Ref. .