KEYWORDS: Fluorescence lifetime imaging, Data modeling, Data acquisition, Classification systems, Tissues, Clinical research, Animal model studies, Library classification systems, Diagnostics, Current controlled current source
The progression of atherosclerosis in coronary vessels involves distinct pathological changes in the vessel wall. These changes manifest in the formation of a variety of plaque sub-types. The ability to detect and distinguish these plaques, especially thin-cap fibroatheromas (TCFA) may be relevant for guiding percutaneous coronary intervention as well as investigating new therapeutics. In this work we demonstrate the ability of fluorescence lifetime imaging (FLIm) derived parameters (lifetime values from sub-bands 390/40 nm, 452/45 nm and 542/50 nm respectively) for generating classification maps for identifying eight different atherosclerotic plaque sub-types in ex vivo human coronary vessels. The classification was performed using a support vector machine based classifier that was built from data gathered from sixteen coronary vessels in a previous study. This classifier was validated in the current study using an independent set of FLIm data acquired from four additional coronary vessels with a new rotational FLIm system. Classification maps were compared to co-registered histological data. Results show that the classification maps allow identification of the eight different plaque sub-types despite the fact that new data was gathered with a different FLIm system. Regions with diffuse intimal thickening (n=10), fibrotic tissue (n=2) and thick-cap fibroatheroma (n=1) were correctly identified on the classification map. The ability to identify different plaque types using FLIm data alone may serve as a powerful clinical and research tool for studying atherosclerosis in animal models as well as in humans.
Raman spectroscopy has been proven to have tremendous potential as biomedical analytical tool for spectroscopic disease diagnostics. The use of fiberoptic coupled Raman spectroscopy systems can enable in-vivo characterization of suspicious lesions. However, Raman spectroscopy has the drawback of rather long acquisition times of several hundreds of milliseconds which makes scanning of larger regions quite challenging. By combining Raman spectroscopy with a fast imaging technique this problem can be alleviate in part. Fluorescence lifetime imaging (FLIm) offers a great potential for such a combination. FLIm can allow for fast tissue area pre-segmentation and location of the points for Raman spectra acquisition. Here, we introduce an optical fiber probe combining FLIm and Raman spectroscopy with an outer diameter of 2 mm. Fluorescence is generated via excitation with a fiber laser at 355 nm. The fluorescence emission is spectrally resolved using a custom-made wavelength-selection module (WSM). The Raman excitation power at 785 nm was set to 50 mW for the in-vivo measurements to prevent sample drying. The lateral probe resolution was determined to be <250 μm for both modalities. This value was taken as step size for several raster scans of different tissue types which were conducted to show the overlap of both modalities under realistic conditions. Finally the probe was used for in vivo raster scans of a rat’s brain and subsequently to acquire FLIm guided Raman spectra of several tissues in and around the craniotomy.
During breast conserving surgery (BCS), which is the preferred approach to treat most early stage breast cancers, the
surgeon attempts to excise the tumor volume, surrounded by thin margin of normal tissue. The intra-operative
assessment of cancerous areas is a challenging procedure, with the surgeon usually relying on visual or tactile guidance.
This study evaluates whether time-resolved fluorescence spectroscopy (TRFS) presents the potential to address this
problem. Point TRFS measurements were obtained from 19 fresh tissue slices (7 patients) and parameters that
characterize the transient signals were quantified via constrained least squares deconvolution scheme. Fibrotic tissue
(FT, n=69), adipose tissue (AT, n=76), and invasive ductal carcinoma (IDC, n=27) were identified in histology and
univariate statistical analysis, followed by multi-comparison test, was applied to the corresponding lifetime data.
Significant differentiation between the three tissue types exists at 390 nm and 500 nm bands. The average lifetime is
3.23±0.74 ns for AT, 4.21±0.83 ns for FT and 4.71±0.35 ns (p<0.05) for IDC at 390 nm. Due to the smaller contribution
of collagen in AT the average lifetime value is different from FT and IDC. Additionally, although intensity
measurements do not show difference between FT and IDC, lifetime can distinguish them. Similarly, in 500 nm these
values are 7.01±1.08 ns, 5.43±1.05 ns and 4.39±0.88 ns correspondingly (p<0.05) and this contrast is due to
differentiation in retinol or flavins relative concentration, mostly contributing to AT. Results demonstrate the potential of
TRFS to intra-operatively characterize BCS breast excised tissue in real-time and assess tumor margins.
The translation of engineered tissues into clinic requires robust monitoring of tissue development, both in vitro and in
vivo. Traditional methods are destructive, time- and cost- inefficient, and do not allow time-lapse measurements from
the same sample or animal. This study reports on the ability of time-resolved fluorescence and ultrasound
measurements for non-destructive characterization of explanted tissue engineered vascular grafts. Results show that
TRFS and FLIm are able to assess alterations in luminal composition namely elastin, collagen and cellular content via
changes in fluorescence lifetime values between normal and grafted tissue. These observations are complemented by
structural changes observed in UBM pertaining to graft integration and neo-intimal and neo-medial thickening. These
results encourage the future application of a catheter-based technique that combines these imaging modalities for nondestructive
characterization of vascular grafts in vivo.
Quantitative and qualitative evaluations of structure and composition are important in monitoring development of engineered vascular tissue both in vitro and in vivo. Destructive techniques are an obstacle for performing time-lapse analyses from a single sample or animal. This study demonstrates the ability of time-resolved fluorescence spectroscopy (TRFS) and ultrasound backscatter microscopy (UBM), as nondestructive and synergistic techniques, for compositional and morphological analyses of tissue grafts, respectively. UBM images and integrated backscatter coefficients demonstrate the ability to visualize and quantify postimplantation changes in vascular graft biomaterials such as loss of the external elastic lamina and intimal/medial thickening over the grafted region as well as graft integration with the surrounding tissue. TRFS results show significant changes in spectra, average lifetime, and fluorescence decay parameters owing to changes in collagen, elastin, and cellular content between normal and grafted tissue regions. These results lay the foundation for the application of a catheter-based technique for in vivo evaluation of vascular grafts using TRFS and UBM.
We report the development and validation of a hybrid intravascular diagnostic system combining multispectral fluorescence lifetime imaging (FLIm) and intravascular ultrasound (IVUS) for cardiovascular imaging applications. A prototype FLIm system based on fluorescence pulse sampling technique providing information on artery biochemical composition was integrated with a commercial IVUS system providing information on artery morphology. A customized 3-Fr bimodal catheter combining a rotational side-view fiberoptic and a 40-MHz IVUS transducer was constructed for sequential helical scanning (rotation and pullback) of tubular structures. Validation of this bimodal approach was conducted in pig heart coronary arteries. Spatial resolution, fluorescence detection efficiency, pulse broadening effect, and lifetime measurement variability of the FLIm system were systematically evaluated. Current results show that this system is capable of temporarily resolving the fluorescence emission simultaneously in multiple spectral channels in a single pullback sequence. Accurate measurements of fluorescence decay characteristics from arterial segments can be obtained rapidly (e.g., 20 mm in 5 s), and accurate co-registration of fluorescence and ultrasound features can be achieved. The current finding demonstrates the compatibility of FLIm instrumentation with in vivo clinical investigations and its potential to complement conventional IVUS during catheterization procedures.
The risk of atherosclerosis plaque rupture cannot be assessed by the current imaging systems and thus new multi-modal
technologies are under investigation. This includes combining a new fluorescence lifetime imaging (FLIm) technique,
which is sensitive to plaque biochemical features, with conventional intravascular ultrasound (IVUS), which provides
information on plaque morphology. In this study we present an automated method allowing for the co-registration of
imaging data acquired based on these two techniques. Intraluminal studies were conducted in ex-vivo segments of human
coronaries with a multimodal catheter integrating a commercial IVUS (40 MHz) and a rotational side-viewing fiber
based multispectral FLIm system (355 nm excitation, 390±20, 452±22 and 542±25 nm acquisition wavelengths). The proposed method relies on the lumen/intima boundary extraction from the IVUS polar images. Image restoration is applied for the noise reduction and edge enhancement, while gray-scale peak tracing over the A-lines of the IVUS polar images is applied for the lumen boundary extraction. The detection of the guide-wire artifact is used for the angular
registration between FLIm and IVUS data, after which the lifetime values can be mapped onto the segmented
lumen/intima interface. The segmentation accuracy has been assessed against manual tracings, providing 0.120±0.054
mm mean Hausdorff distance. This method makes the bi-modal FLIm and IVUS approach feasible for comprehensive
intravascular diagnostic by providing co-registered biochemical and morphological information about atherosclerotic
plaques.
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