Autofluorescence lifetime spectroscopy is a promising non-invasive label-free tool for characterization of biological tissues and shows potential to report structural and biochemical alterations in tissue owing to pathological transformations. In particular, when combined with fiber-optic based instruments, autofluorescence lifetime measurements can enhance intraoperative diagnosis and provide guidance in surgical procedures. We investigate the potential of a fiber-optic based multi-spectral time-resolved fluorescence spectroscopy instrument to characterize the autofluorescence fingerprint associated with histologic, morphologic and metabolic changes in tissue that can provide real-time contrast between healthy and tumor regions in vivo and guide clinicians during resection of diseased areas during transoral robotic surgery. To provide immediate feedback to the surgeons, we employ tracking of an aiming beam that co-registers our point measurements with the robot camera images and allows visualization of the surgical area augmented with autofluorescence lifetime data in the surgeon’s console in real-time. For each patient, autofluorescence lifetime measurements were acquired from normal, diseased and surgically altered tissue, both in vivo (pre- and post-resection) and ex vivo. Initial results indicate tumor and normal regions can be distinguished based on changes in lifetime parameters measured in vivo, when the tumor is located superficially. In particular, results show that autofluorescence lifetime of tumor is shorter than that of normal tissue (p < 0.05, n = 3). If clinical diagnostic efficacy is demonstrated throughout this on-going study, we believe that this method has the potential to become a valuable tool for real-time intraoperative diagnosis and guidance during transoral robot assisted cancer removal interventions.
Multi-Spectral Time-Resolved Fluorescence Spectroscopy (ms-TRFS) can provide label-free real-time feedback on tissue composition and pathology during surgical procedures by resolving the fluorescence decay dynamics of the tissue. Recently, an ms-TRFS system has been developed in our group, allowing for either point-spectroscopy fluorescence lifetime measurements or dynamic raster tissue scanning by merging a 450 nm aiming beam with the pulsed fluorescence excitation light in a single fiber collection. In order to facilitate an augmented real-time display of fluorescence decay parameters, the lifetime values are back projected to the white light video. The goal of this study is to develop a 3D real-time surface reconstruction aiming for a comprehensive visualization of the decay parameters and providing an enhanced navigation for the surgeon. Using a stereo camera setup, we use a combination of image feature matching and aiming beam stereo segmentation to establish a 3D surface model of the decay parameters. After camera calibration, texture-related features are extracted for both camera images and matched providing a rough estimation of the surface. During the raster scanning, the rough estimation is successively refined in real-time by tracking the aiming beam positions using an advanced segmentation algorithm. The method is evaluated for excised breast tissue specimens showing a high precision and running in real-time with approximately 20 frames per second. The proposed method shows promising potential for intraoperative navigation, i.e. tumor margin assessment. Furthermore, it provides the basis for registering the fluorescence lifetime maps to the tissue surface adapting it to possible tissue deformations.
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.
Fluorescence lifetime imaging has been shown to be a robust technique for biochemical and functional characterization
of tissues and to present great potential for intraoperative tissue diagnosis and guidance of surgical procedures. We
report a technique for real-time mapping of fluorescence parameters (i.e. lifetime values) onto the location from where
the fluorescence measurements were taken. This is achieved by merging a 450 nm aiming beam generated by a diode
laser with the excitation light in a single delivery/collection fiber and by continuously imaging the region of interest with
a color CMOS camera. The interrogated locations are then extracted from the acquired frames via color-based
segmentation of the aiming beam. Assuming a Gaussian profile of the imaged aiming beam, the segmentation results are
fitted to ellipses that are dynamically scaled at the full width of three automatically estimated thresholds (50%, 75%,
90%) of the Gaussian distribution's maximum value. This enables the dynamic augmentation of the white-light video
frames with the corresponding fluorescence decay parameters. A fluorescence phantom and fresh tissue samples were
used to evaluate this method with motorized and hand-held scanning measurements. At 640x512 pixels resolution the
area of interest augmented with fluorescence decay parameters can be imaged at an average 34 frames per second. The
developed method has the potential to become a valuable tool for real-time display of optical spectroscopy data during
continuous scanning applications that subsequently can be used for tissue characterization and diagnosis.
We report a scanning imaging system that enables high speed multispectral fluorescence lifetime imaging (FLIm) of
coronary arteries. This system combines a custom low profile (3 Fr) imaging catheter using a 200 μm core side viewing
UV-grade silica fiber optic, an acquisition system able to measure fluorescence decays over four spectral bands at 20
kHz and a fast data analysis and display module. In vivo use of the system has been optimized, with particular emphasis
on clearing blood from the optical pathway. A short acquisition time (5 seconds for a 20 mm long coronary segment)
enabled data acquisition during a bolus saline solution injection through the 7 Fr catheter guide. The injection parameters
were precisely controlled using a power injector and optimized to provide good image quality while limiting the bolus
injection duration and volume (12 cc/s, 80 cc total volume). The ability of the system to acquire data in vivo was
validated in healthy swine by imaging different sections of the left anterior descending (LAD) coronary. A stent coated
with fluorescent markers was placed in the LAD and imaged, demonstrating the ability of the system to discriminate in
vivo different fluorescent features and structures from the vessel background fluorescence using spectral and lifetime
information. Intensity en face images over the four bands of the instrument were available within seconds whereas
lifetime images were computed in 2 minutes, providing efficient feedback during the procedure. This successful
demonstration of FLIm in coronaries enables future study of atherosclerotic cardiovascular diseases.
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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