This study demonstrates the novel application of a fiber-based point-spectroscopy TRFS system to record fluorescence data in a continuous helical scanning (radial) manner across multiple spectral emission bands; and to accurately reconstruct and resolve fluorescence lifetimes of fluorescent targets located on tubular structures (e.g., luminal surface of arterial wall). The performance of the scanning-TRFS system to create FLIM images (including the image pixel size, data acquisition speed, SNR, and accuracy of lifetime values) were found to depend on a range of the experimental parameters such as laser repetition rate, average number of fluorescence pulses to improve SNR, beam size, scanning speed, lumen diameter, bandwidth and speed of the digitizer, response time and sensitivity of the detector. These are discussed in the following. Based on current results, optimized imaging parameters can be determined for specific application in subsequent studies.
As anticipated, the evaluation of the effect of the scanning speed on the pixel size showed that the lower scanning speed results in smaller pixel size (0.04 mm pixel size with 0.1 mm/s versus 0.4 mm with 1 mm/s) and thus improved image quality. However, this is at the expense of increased data acquisition time ranging from a few to several minutes. For the scanning-TRFS system described here using the digitizer’s build in FastFrame data acquisition mode, the laser repetition rate is the major limiting factor in improving the scanning speed while preserving the image quality. The current averaged acquisition time per pixel is ~0.4 s, which includes the time needed for averaging of 6 consecutive pulses at 30 Hz, data processing, displaying, and storing data. Currently, it takes 1 to 15 minutes to record a FLIM image, depending on the size of scanning area. The acquisition time can be significantly reduced by using a fast repetition rate laser. We anticipate that use of pulse rate at 10 kHz combined with the same FastFrame data acquisition mode can reduce this scanning time from minutes to a few seconds.
The spatial resolution of the scanning TRFS system was mainly determined by the size of the optical beam at the tissue/sample surface and light excitation-collection geometry (fiber-to-tissue distance). As demonstrated by the experiments concerning the effect of vessel diameter on the ability to resolve two fluorophores in the fluorescence image (), the increased working distance due to a large lumen diameter resulted in not only a larger pixel size (0.07 mm for 2 mm vessel versus 0.21 mm for 6 mm vessel) but also reduced spatial resolution. The current system resolution, which was characterized as ~250 µm by measuring the point spread function (), can be improved by decreasing the numerical aperture of the fiber optic or by increasing the refractive index of the media in the biological application in which the laser beam travels through. Inherently, applications in an intravascular settings will benefit from the latter since the spatial resolution could be improved in the water/saline environment (n = ~1.3) compared to air (n = 1.0).
An important feature of the scanning-TRFS system described here is its ability to simultaneously resolve the fluorescence intensity decay in multiple spectral bands. The current system allows for spectral separation of all fluorophores used in the validation tests ( and ). Each of the fluorescent structures (e.g., ) can be clearly observed in distinct spectral channels in various intensity scales. Moreover, overlap in spectral emission of the fluorescent structures can be further delineated using time-resolved measurements (e.g., C120 and C1). Overall, current results demonstrate that this system is capable of resolving the fluorescence decay of most fluorescent components in biological tissue with lifetimes ranging from 2 ns to 12 ns in a broad spectral band ranging from about 370 nm to 650 nm. While certain spectral bands were used in this study, the modular design of current instrument enables selection of any spectral band of interest by simply replacing the set of dichroic filters and band-pass filters within the wavelength selection module ().
One of the goals in the development of the scanning-TRFS system is to ultimately apply such a system in clinical cardiovascular diagnosis. This study not only has demonstrated that such approach can facilitate robust and continuous TRFS data acquisition from the arterial lumen, but also the identification of fluorescent molecular structures with distinct fluorescence lifetime against the autofluorescence emission of normal arterial wall. For atherosclerotic plaque characterization and diagnosis, it is important to differentiate molecular composition within the vessel wall (i.e., elastin, collagen and lipids) that are associate with distinct plaque pathological features. The hybrid tissue phantom, including pig aorta, fluorophore dyes, and a stent, enabled evaluation of a few conditions that are useful for future practical implementation of this technique. For example, the tests conducted in this phantom showed that the scanning-TRFS system is sensitive enough to resolve the autofluorescence emission of the normal arterial wall. While the fluorescent dyes used to generate fluorescent targets cannot mimic entirely the fluorescence emission of lipid-rich atherosclerotic plaques, they allowed for testing whether the emission of molecular structures with different decay dynamics than the normal arterial tissue can be resolved. The reconstructed fluorescence lifetime revealed that fluorescent targets with shorter lifetime values (~3 ns) than the aorta autofluorescence (~5 ns) can be distinguished from normal aortic tissue (). Since lipid-rich plaques were shown to exhibit faster decay dynamics than normal arterial wall rich in elastin and collagen [8
], current results demonstrate that the scanning-TRFS has the potential to localize such plaques that are associated with critical cardiovascular events. The use of the stent allowed observation of the scanning system to resolve nonfluorescent structures within the vessel wall.
Moreover, the fiber optic-based rotational scanning system allows for further integration of this device coupled to other intravascular techniques such as intravascular ultrasound (IVUS) [25
]. Such bi-modal system can provide a simultaneous characterization of both biochemical and structural characteristics of the atherosclerotic plaques. A few approaches for coupling these two modalities were described in our earlier publications [11
]. When applied synchronously, IVUS not only can provide structural information on the vessel wall but also information on the lumen diameter and the position of the optical fiber relative to the vessel wall. Based on the information on the lumen diameter acquired by IVUS, the pixel size and fluorescence signal can be corrected to reconstruct the FLIM images in cases where the size of the lumen in not uniform or the fiber is not at the centered in the lumen. The fluorescence intensity variation due to changes distance between the tip of the fiber and vessel wall is less of a concern for time-resolved fluorescence measurements, as the information derived from these measurements is primarily based on the analysis of the fluorescence decay dynamics rather than absolute intensity. Developments in this direction are currently pursued by our group.
In conclusion, we demonstrated the feasibility of a prototype scanning-TRFS system with fiber optic for performing a helical scan to acquire intraluminal FLIM data. The scanning system was validated by recording the fluorescence signals in a continuous data acquisition sequence using in vitro
physical phantoms and a hybrid ex vivo
pig artery phantom. The performance of the scanning TRFS system was characterized with multiple experimental settings including different scanning speeds and lumen diameters in a vessel phantom. The robust fluorescence lifetime retrievability during scanning-TRFS measurements suggests a great potential for application of this scanning system for intravascular cardiovascular diagnosis. The use of high repetition laser devices (e.g., Fianium fiber laser with repetition rate up to 20 MHz) along with advancement of new solutions for continuously removing the blood from the optical pathway as recently reported [26
] is anticipated to facilitate development of the next generation of scanning-TRFS systems and in vivo
FLIM-based characterization of plaque cap composition during intravascular interventions.