Dinglong Ma

Design and evaluation of a device for fast multispectral time-resolved fluorescence spectroscopy and imaging

The application of time-resolved fluorescence spectroscopy (TRFS) to in vivo tissue diagnosis requires a method for fast acquisition of fluorescence decay profiles in multiple spectral bands. This study focusses on development of a clinically compatible fiber-optic based multispectral TRFS (ms-TRFS) system together with validation of its accuracy and precision for fluorescence lifetime measurements. It also presents the expansion of this technique into an imaging spectroscopy method. A tandem array of dichroic beamsplitters and filters was used to record TRFS decay profiles at four distinct spectral bands where biological tissue typically presents fluorescence emission maxima, namely, 390, 452, 542, and 629 nm. Each emission channel was temporally separated by using transmission delays through 200 μm diameter multimode optical fibers of 1, 10, 19, and 28 m lengths. A Laguerre-expansion deconvolution algorithm was used to compensate for modal dispersion inherent to large diameter optical fibers and the finite bandwidth of detectors and digitizers. The system was found to be highly efficient and fast requiring a few nano-Joule of laser pulse energy and <1 ms per point measurement, respectively, for the detection of tissue autofluorescent components. Organic and biological chromophores with lifetimes that spanned a 0.8-7 ns range were used for system validation, and the measured lifetimes from the organic fluorophores deviated by less than 10% from values reported in the literature. Multi-spectral lifetime images of organic dye solutions contained in glass capillary tubes were recorded by raster scanning the single fiber probe in a 2D plane to validate the system as an imaging tool. The lifetime measurement variability was measured indicating that the system provides reproducible results with a standard deviation smaller than 50 ps. The ms-TRFS is a compact apparatus that makes possible the fast, accurate, and precise multispectral time-resolved fluorescence lifetime measurements of low quantum efficiency sub-nanosecond fluorophores.

Combined fiber probe for fluorescence lifetime and Raman spectroscopy

AbstractIn this contribution we present a dual modality fiber optic probe combining fluorescence lifetime imaging (FLIm) and Raman spectroscopy for in vivo endoscopic applications. The presented multi-spectroscopy probe enables efficient excitation and collection of fluorescence lifetime signals for FLIm in the UV/visible wavelength region, as well as of Raman spectra in the near-IR for simultaneous Raman/FLIm imaging. The probe was characterized in terms of its lateral resolution and distance dependency of the Raman and FLIm signals. In addition, the feasibility of the probe for in vivo FLIm and Raman spectral characterization of tissue was demonstrated. Graphical AbstractAn image comparison between FLIm and Raman spectroscopy acquired with the bimodal probe onseveral tissue samples

Real-Time Visualization of Tissue Surface Biochemical Features Derived From Fluorescence Lifetime Measurements

Fiber based fluorescence lifetime imaging has shown great potential for intraoperative diagnosis and guidance of surgical procedures. Here we describe a novel method addressing a significant challenge for the practical implementation of this technique, i.e., the real-time display of the quantified biochemical or functional tissue properties superimposed on the interrogated area. Specifically, an aiming beam (450 nm) generated by a continuous-wave laser beam was merged with the pulsed fluorescence excitation light in a single delivery/collection fiber and then imaged and segmented using a color-based algorithm. We demonstrate that this approach enables continuous delineation of the interrogated location and dynamic augmentation of the acquired frames with the corresponding fluorescence decay parameters. The method was evaluated on a fluorescence phantom and fresh tissue samples. Current results demonstrate that 34 frames per second can be achieved for augmenting videos of 640 × 512 pixels resolution. Also we show that the spatial resolution of the fluorescence lifetime map depends on the tissue optical properties, the scanning speed, and the frame rate. The dice similarity coefficient between the fluorescence phantom and the reconstructed maps was estimated to be as high as 93%. The reported method could become a valuable tool for augmenting the surgeons field of view with diagnostic information derived from the analysis of fluorescence lifetime data in real-time using handheld, automated, or endoscopic scanning systems. Current method provides also a means for maintaining the tissue light exposure within safety limits. This study provides a framework for using an aiming beam with other point spectroscopy applications.

Technique for real-time tissue characterization based on scanning multispectral fluorescence lifetime spectroscopy (ms-TRFS)

We report a novel technique for continuous acquisition, processing and display of fluorescence lifetimes enabling real-time tissue diagnosis through a single hand held or biopsy fiber-optic probe. A scanning multispectral time-resolved fluorescence spectroscopy (ms-TRFS) with self-adjustable photon detection range was developed to account for the dynamic changes of fluorescence intensity typically encountered in clinical application. A fast algorithm was implemented in the ms-TRFS software platform, providing up to 15 Hz continuous display of fluorescence lifetime values. Potential applications of this technique, including biopsy guidance, and surgical margins delineation were demonstrated in proof-of-concept experiments. Current results showed accurate display of fluorescence lifetimes values and discrimination of distinct fluorescence markers and tissue types in real-time (< 100 ms per data point).

Comparing Raman and fluorescence lifetime spectroscopy from human atherosclerotic lesions using a bimodal probe.

Fluorescence lifetime imaging (FLIm) and Raman spectroscopy are two promising methods to support morphological intravascular imaging techniques with chemical contrast. Both approaches are complementary and may also be used in combination with OCT/IVUS to add chemical specificity to these morphologic intravascular imaging modalities. In this contribution, both modalities were simultaneously acquired from two human coronary specimens using a bimodal probe. A previously trained SVM model was used to interpret the fluorescence lifetime data; integrated band intensities displayed in RGB false color images were used to interpret the Raman data. Both modalities demonstrate unique strengths and weaknesses and these will be discussed in comparison to histologic analyses from the two coronary arteries imaged.

Fluorescence Lifetime Imaging and Intravascular Ultrasound: Co-Registration Study Using Ex Vivo Human Coronaries

Fluorescence lifetime imaging (FLIM) has demonstrated potential for robust assessment of atherosclerotic plaques biochemical composition and for complementing conventional intravascular ultrasound (IVUS), which provides information on plaque morphology. The success of such a bi-modal imaging modality depends on accurate segmentation of the IVUS images and proper angular registration between these two modalities. This paper reports a novel IVUS segmentation methodology addressing this issue. The image preprocessing consisted of denoising, using the Wiener filter, followed by image smoothing, implemented through the application of the alternating sequential filter on the edge separability metric images. Extraction of the lumen/intima and media/adventitia boundaries was achieved by tracing the gray-scale peaks over the A-lines of the IVUS preprocessed images. Cubic spline interpolation, in both cross-sectional and longitudinal directions, ensured boundary smoothness and continuity. The detection of the guide-wire artifact in both modalities is used for angular registration. Intraluminal studies were conducted in 13 ex vivo segments of human coronaries. The IVUS segmentation accuracy was assessed against independent manual tracings, providing 91.82% sensitivity and 97.55% specificity. The proposed methodology makes the bi-modal FLIM and IVUS approach feasible for comprehensive intravascular diagnosis by providing co-registered biochemical and morphological information of atherosclerotic plaques.

In-vivo validation of fluorescence lifetime imaging (FLIm) of coronary arteries in swine

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.

In vivo label-free structural and biochemical imaging of coronary arteries using an integrated ultrasound and multispectral fluorescence lifetime catheter system

Existing clinical intravascular imaging modalities are not capable of accurate detection of critical plaque pathophysiology in the coronary arteries. This study reports the first intravascular catheter combining intravascular ultrasound (IVUS) with multispectral fluorescence lifetime imaging (FLIm) that enables label-free simultaneous assessment of morphological and biochemical features of coronary vessels in vivo. A 3.7 Fr catheter with a fiber-optic channel was constructed based on a 40 MHz clinical IVUS catheter. The ability to safely acquire co-registered FLIm-IVUS data in vivo using Dextran40 solution flushing was demonstrated in swine coronary arteries. FLIm parameters from the arterial wall were consistent with the emission of fluorophores present in healthy arterial wall (collagen, elastin). Additionally, structural and biochemical features from atherosclerotic lesions were acquired in ex vivo human coronary samples and corroborated with histological findings. Current results show that FLIm parameters linked to the amount of structural proteins (e.g. collagen, elastin) and lipids (e.g. foam cells, extracellular lipids) in the first 200 μm of the intima provide important biochemical information that can supplement IVUS data for a comprehensive assessment of plaques pathophysiology. The unique FLIm-IVUS system evaluated here has the potential to provide a comprehensive insight into atherosclerotic lesion formation, diagnostics and response to therapy.

Bi-modal imaging of atherosclerotic plaques: Automated method for co-registration between fluorescence lifetime imaging and intravascular ultrasound data

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.

Electrocautery effects on fluorescence lifetime measurements: An in vivo study in the oral cavity

Abstract Tumor removal typically involves electrocautery, but no studies to date have quantified the effect of electrocautery on fluorescence emission. Electrocautery was applied to N=4 locations of the oral cavity and striated leg muscle of a live Yorkshire pig. Autofluorescence of cauterized tissues and surrounding regions was measured at distinct time points up to 120 minutes following cauterization. The fluorescence lifetime was spectrally resolved in four spectral detection channels that maximized the signal emanating from endogenous fluorophores of interest. The autofluorescence emission (355 nm excitation) was temporally resolved using a high-speed digitizer; resulting fluorescence decay characteristics were retrieved using the Laguerre deconvolution technique. Histology was performed and co-registered with the autofluorescence data. Results show that cauterized tissue presents a distinct autofluorescence signature from surrounding regions immediately after cauterization. Differences become less evident with time. The autofluorescence-derived parameters suggest altered metabolism in peripheral regions compared to the region of maximal damage. Within the time-frame of this study, tissues investigated show variable degrees of recovery from the effects of electrocautery that can be monitored by changes in fluorescence lifetime characteristics. Our findings suggest delineation of pathologic conditions could be affected by tissue cauterization and that future studies in this area will be necessary.