Multispectral fluorescence lifetime imaging device with a silicon avalanche photodetector.

We report the design, development, and characterization of a novel multi-spectral fluorescence lifetime measurement device incorporating solid-state detectors and automated gain control. For every excitation pulse (∼1 µJ, 600 ps), this device records complete fluorescence decay from multiple spectral channels simultaneously within microseconds, using a dedicated UV enhanced avalanche photodetector and analog to digital convert (2.5 GS/s) in each channel. Fast (<2 ms) channel-wise dynamic range adjustment maximizes the signal-to-noise ratio. Fluorophores with known lifetime ranging from 0.5-6.0 ns were used to demonstrate the device accuracy. Current results show the clear benefits of this device compared to existing devices employing microchannel-plate photomultiplier tubes. This is demonstrated by 5-fold reduction of lifetime measurement variability in identical conditions, independent gain adjustment in each spectral band, and 4-times faster imaging speed. The use of solid-state detectors will also facilitate future improved performance and miniaturization of the instrument.

Fiber-based platform for synchronous imaging of endogenous and exogenous fluorescence of biological tissue.

Endogenous and exogenous fluorescence emission from biological samples encodes complementary information. Here we report, to the best of our knowledge, the first results from an optical imaging platform with interleaved excitation and detection of exogenous and endogenous fluorescence from tissue samples using a single flexible multimode fiber that delivers the excitation beam and collects the emitted light. A custom-built reflective optical chopper wheel with synchronized rotation temporally multiplexes an autofluorescence lifetime imaging apparatus with an intensity-based fluorescence module tailored to imaging green fluorescent protein. We demonstrate the functionality of such platform imaging dyes of varying fluorescence signatures and resolving cellularized areas on bio-engineered tissue constructs.

Multiscale, multispectral fluorescence lifetime imaging using a double-clad fiber.

Fiber-based imaging of tissue autofluorescence using ultraviolet (UV) excitation is a highly flexible tool used to probe structure and composition. In this Letter, we report, to the best of our knowledge, the first results from a single-fiber imaging system employing a custom double-clad fiber to acquire multispectral fluorescence lifetime images at two distinct spatial resolutions. We characterize the lateral point spread function and fluorescent background of the system and show how enhanced resolution can identify features such as trabeculae in ex vivo murine bone 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 surgeon's 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).

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.

Rotational multispectral fluorescence lifetime imaging and intravascular ultrasound: bimodal system for intravascular applications.

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.

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.

Development of a new pulsed source for photoacoustic imaging based on aperiodically poled lithium niobate.

We present the development of a source of deep-red radiation for photoacoustic imaging. This source, which is based on two cascaded wavelength conversion processes in aperiodically poled lithium niobate, emits 10 nanosecond pulses of over 500 µJ at 710 nm. Photoacoustic images were obtained from phantoms designed to mimic the optical and acoustic properties of oral tissue. Results indicate this device is a viable source of optical pulses for photoacoustic applications.

Multispectral fluorescence lifetime imaging system for intravascular diagnostics with ultrasound guidance: in vivo validation in swine arteries.

Fluorescence lifetime technique has demonstrated potential for analysis of atherosclerotic lesions and for complementing existing intravascular imaging modalities such as intravascular ultrasound (IVUS) in identifying lesions at high risk of rupture. This study presents a multimodal catheter system integrating a 40 MHz commercial IVUS and fluorescence lifetime imaging (FLIm) using fast helical motion scanning (400 rpm, 0.75 mm/s), able to acquire in vivo in pulsatile blood flow the autofluorescence emission of arterial vessels with high precision (5.08 ± 0.26 ns mean average lifetime over 13 scans). Co-registered FLIm and IVUS data allowed 3D visualization of both biochemical and morphological vessel properties. Current study supports the development of clinically compatible intravascular diagnostic system integrating FLIm and demonstrates, to our knowledge, the first in vivo intravascular application of a fluorescence lifetime imaging technique.

Multimodal in vivo imaging of oral cancer using fluorescence lifetime, photoacoustic and ultrasound techniques.

This work reports a multimodal system for label-free tissue diagnosis combining fluorescence lifetime imaging (FLIm), ultrasound backscatter microscopy (UBM), and photoacoustic imaging (PAI). This system provides complementary biochemical, structural and functional features allowing for enhanced in vivo detection of oral carcinoma. Results from a hamster oral carcinoma model (normal, precancer and carcinoma) are presented demonstrating the ability of FLIm to delineate biochemical composition at the tissue surface, UBM and related radiofrequency parameters to identify disruptions in the tissue microarchitecture and PAI to map optical absorption associated with specific tissue morphology and physiology.

Design, construction, and validation of a rotary multifunctional intravascular diagnostic catheter combining multispectral fluorescence lifetime imaging and intravascular ultrasound.

We report the development and validation of an intravascular rotary catheter for bimodal interrogation of arterial pathologies. This is based on a point-spectroscopy scanning time-resolved fluorescence spectroscopy technique enabling reconstruction of fluorescence lifetime images (FLIm) and providing information on arterial intima composition and intravascular ultrasound (IVUS) providing information on arterial wall morphology. The catheter design allows for independent rotation of the ultrasonic and optical channels within an 8 Fr outer diameter catheter sheath and integrates a low volume flushing channel for blood removal in the optical pathways. In the current configuration, the two channels consist of (a) a standard 3 Fr IVUS catheter with single element transducer (40 MHz) and (b) a side-viewing fiber optic (400 µm core). Experiments conducted in tissue phantoms showed the ability of the catheter to operate in an intraluminal setting and to generate coregistered FLIm and IVUS in one pull-back scan. Current results demonstrate the feasibility of the catheter for simultaneous bimodal interrogation of arterial lumen and for generation of robust fluorescence lifetime data under IVUS guidance. These results facilitate further development of a FLIm-IVUS technique for intravascular diagnosis of atherosclerotic cardiovascular diseases including vulnerable plaques.

Structural origins of chiral second-order optical nonlinearity in collagen: amide I band.

The molecular basis of nonlinear optical (NLO) chiral effects in the amide I region of type I collagen was investigated using sum-frequency generation vibrational spectroscopy; chiral and achiral tensor elements were separated using different input/output beam polarization conditions. Spectra were obtained from native rat tail tendon (RTT) collagen and from cholesteric liquid crystal-like (LC) type I collagen films. Although RTT and LC collagen both possess long-range order, LC collagen lacks the complex hierarchical organization of RTT collagen. Their spectra were compared to assess the role of such organization in NLO chirality. No significant differences were observed between RTT and LC with respect to chiral or achiral spectra. These findings suggest that amide I NLO chiral effects in type I collagen assemblies arise predominantly from the chiral organization of amide chromophores within individual collagen molecules, rather than from supramolecular structures. The study suggests that sum-frequency generation vibrational spectroscopy may be uniquely valuable in exploring fundamental aspects of chiral nonlinearity in complex macromolecular structures.

In vivo validation of a bimodal technique combining time-resolved fluorescence spectroscopy and ultrasonic backscatter microscopy for diagnosis of oral carcinoma.

Tissue diagnostic features generated by a bimodal technique integrating scanning time-resolved fluorescence spectroscopy (TRFS) and ultrasonic backscatter microscopy (UBM) are investigated in an in vivo hamster oral carcinoma model. Tissue fluorescence is excited by a pulsed nitrogen laser and spectrally and temporally resolved using a set of filters/dichroic mirrors and a fast digitizer, respectively. A 41-MHz focused transducer (37-µm axial, 65-µm lateral resolution) is used for UBM scanning. Representative lesions of the different stages of carcinogenesis show that fluorescence characteristics complement ultrasonic features, and both correlate with histological findings. These results demonstrate that TRFS-UBM provide a wealth of co-registered, complementary data concerning tissue composition and structure as it relates to disease status. The direct co-registration of the TRFS data (sensitive to surface molecular changes) with the UBM data (sensitive to cross-sectional structural changes and depth of tumor invasion) is expected to play an important role in pre-operative diagnosis and intra-operative determination of tumor margins.

Multispectral scanning time-resolved fluorescence spectroscopy (TRFS) technique for intravascular diagnosis.

This study describes a scanning time-resolved fluorescence spectroscopy (TRFS) system designed to continuously acquire fluorescence emission and to reconstruct fluorescence lifetime images (FLIM) from a luminal surface by using a catheter-based optical probe with rotary joint and pull-back device. The ability of the system to temporally and spectrally resolve the fluorescence emission from tissue was validated using standard dyes and tissue phantoms (e.g., ex vivo pig aorta phantom). Current results demonstrate that this system is capable to reliably resolve the fluorescence emission of multiple fluorophores located in the lumen; and suggest its potential for intravascular detection of distinct biochemical features of atherosclerotic plaques.

Reversible low-light induced photoswitching of crowned spiropyran-DO3A complexed with gadolinium(III) ions.

Photoswitchable spiropyran has been conjugated to the crowned ring system DO3A, which improves its solubility in dipolar and polar media and stabilizes the merocyanine isomer. Adding the lanthanide ion gadolinium(III) to the macrocyclic ring system leads to a photoresponsive magnetic resonance imaging contrast agent that displays an increased spin-lattice relaxation time (T1) upon visible light stimulation. In this work, the photoresponse of this photochromic molecule to weak light illumination using blue and green light emitting diodes was investigated, simulating the emission spectra from bioluminescent enzymes. Photon emission rate of the light emitting diodes was changed, from 1.75 × 10¹6 photons·s⁻¹ to 2.37 × 10¹² photons·s⁻¹. We observed a consistent visible light-induced isomerization of the merocyanine to the spiropyran form with photon fluxes as low as 2.37 × 10¹² photons·s⁻¹ resulting in a relaxivity change of the compound. This demonstrates the potential for use of the described imaging probes in low light level applications such as sensing bioluminescence enzyme activity. The isomerization behavior of gadolinium(III)-ion complexed and non-complexed spiropyran-DO3A was analyzed in water and ethanol solution in response to low light illumination and compared to the emitted photon emission rate from over-expressed Gaussia princeps luciferase.

Two-photon excited fluorescence lifetime measurements through a double-clad photonic crystal fiber for tissue micro-endoscopy.

This paper presents an endoscopic configuration for measurements of tissue autofluorescence using two-photon excitation and time-correlated single photon counting detection through a double-clad photonic crystal fiber (DC-PCF) without pre-chirping of laser pulses. The instrument performance was evaluated by measurements of fluorescent standard dyes, biological fluorophores (collagen and elastin), and tissue specimens (muscle, cartilage, tendon). Current results demonstrate the ability of this system to accurately retrieve the fluorescence decay profile and lifetime of these samples. This simple setup, which offers larger penetration depth than one-photon-based techniques, may be combined with morphology-yielding techniques such as photoacoustic and ultrasound imaging.

Multimodal characterization of compositional, structural and functional features of human atherosclerotic plaques.

Detection of atherosclerotic plaque vulnerability has critical clinical implications for avoiding sudden death in patients with high risk of plaque rupture. We report on multimodality imaging of ex-vivo human carotid plaque samples using a system that integrates fluorescence lifetime imaging (FLIM), ultrasonic backscatter microscopy (UBM), and photoacoustic imaging (PAI). Biochemical composition is differentiated with a high temporal resolution and sensitivity at the surface of the plaque by the FLIM subsystem. 3D microanatomy of the whole plaque is reconstructed by the UBM. Functional imaging associated with optical absorption contrast is evaluated from the PAI component. Simultaneous recordings of the optical, ultrasonic, and photoacoustic data present a wealth of complementary information concerning the plaque composition, structure, and function that are related to plaque vulnerability. This approach is expected to improve our ability to study atherosclerotic plaques. The multimodal system presented here can be translated into a catheter based intraluminal system for future clinical studies.

Sum frequency vibrational spectroscopy: the molecular origins of the optical second-order nonlinearity of collagen.

The molecular origins of second-order nonlinear effects in type I collagen fibrils have been identified with sum-frequency generation vibrational spectroscopy. The dominant contributing molecular groups are: 1), the methylene groups associated with a Fermi resonance between the fundamental symmetric stretch and the bending overtone of methylene; and 2), the carbonyl and peptide groups associated with the amide I band. The noncentrosymmetrically aligned methylene groups are characterized by a distinctive tilt relative to the axis perpendicular to the main axis of the collagen fiber, a conformation producing a strong achiral contribution to the second-order nonlinear effect. In contrast, the stretching vibration of the carbonyl groups associated with the amide I band results in a strong chiral contribution to the optical second-order nonlinear effect. The length scale of these chiral effects ranges from the molecular to the supramolecular.

Quantitative analysis of structural disorder in intervertebral disks using second harmonic generation imaging: comparison with morphometric analysis.

A novel signal processing algorithm for quantifying structural disorder in biological tissue using second harmonic generation (SHG) imaging is described. Both the magnitude and the pattern of disorder in collagenous tissues can be determined with this method. Mathematical models are used to determine the range of disordered states over which the algorithm can be used, because highly disordered biological samples do not generate second harmonic signals. The method is validated by measuring disorder in heated fascicles using SHG and showing that results are significantly correlated with morphometric determination. Applicability of the method to tissue pathology is demonstrated by analysis of a mouse model of intervertebral disk injury. Disks were subjected to tensile or compressive forces in vivo for one week. Structural disorder in the annulus fibrosus was measured by SHG scanning and by standard morphometric analysis. Values for disorder obtained by SHG scanning were significantly correlated with values obtained by morphometry (p<0.001). Quantitation of disorder using SHG offers significant advantages over morphometric determination. Data obtained in this study suggest that this method can be used to discriminate between reversible and irreversible tissue damage.