Sebina Shrestha

A dual-modality optical coherence tomography and fluorescence lifetime imaging microscopy system for simultaneous morphological and biochemical tissue characterization

Most pathological conditions elicit changes in the tissue optical response that may be interrogated by one or more optical imaging modalities. Any single modality typically only furnishes an incomplete picture of the tissue optical response, hence an approach that integrates complementary optical imaging modalities is needed for a more comprehensive non-destructive and minimally-invasive tissue characterization. We have developed a dual-modality system, incorporating optical coherence tomography (OCT) and fluorescence lifetime imaging microscopy (FLIM), that is capable of simultaneously characterizing the 3-D tissue morphology and its biochemical composition. The Fourier domain OCT subsystem, at an 830 nm center wavelength, provided high-resolution morphological volumetric tissue images with an axial and lateral resolution of 7.3 and 13.4 µm, respectively. The multispectral FLIM subsystem, based on a direct pulse-recording approach (upon 355 nm laser excitation), provided two-dimensional superficial maps of the tissue autofluorescence intensity and lifetime at three customizable emission bands with 100 µm lateral resolution. Both subsystems share the same excitation/illumination optical path and are simultaneously raster scanned on the sample to generate coregistered OCT volumes and FLIM images. The developed OCT/FLIM system was capable of a maximum A-line rate of 59 KHz for OCT and a pixel rate of up to 30 KHz for FLIM. The dual-modality system was validated with standard fluorophore solutions and subsequently applied to the characterization of two biological tissue types: postmortem human coronary atherosclerotic plaques, and in vivo normal and cancerous hamster cheek pouch epithelial tissue.

High-speed multispectral fluorescence lifetime imaging implementation for in vivo applications

Fluorescence lifetime imaging microscopy (FLIM) offers a noninvasive approach for characterizing the biochemical composition of biological tissue. In recent years, there has been an increasing interest in the application of multispectral FLIM for medical diagnosis. Central to the clinical translation of FLIM technology is the development of robust, fast, and cost-effective FLIM instrumentation suitable for in vivo tissue imaging. Unfortunately, the predominant multispectral FLIM approaches suffer from limitations that impede the development of high-speed instruments for in vivo applications. We present a cost-effective scanning multispectral FLIM implementation capable of achieving pixel rates on the order of tens of kilohertz, which will facilitate the evaluation of FLIM for in vivo applications.

In Vivo Simultaneous Morphological and Biochemical Optical Imaging of Oral Epithelial Cancer

Early detection of cancer is key to reducing morbidity and mortality. Morphological and chemical biomarkers presage the transition from normal to cancerous tissue. We have developed a noninvasive imaging system incorporating optical coherence tomography (OCT) and fluorescence lifetime imaging microscopy (FLIM) into a single optical system for the first time, in order to acquire both sets of biomarkers. OCT can provide morphological images of tissue with high resolution, while FLIM can provide biochemical tissue maps. Coregistered OCT volumes and FLIM images have been acquired simultaneously in an in vivo hamster cheek pouch model of oral cancer. The OCT images indicate morphological biomarkers for cancer including thickening of the epithelial layer and loss of the layered structure. The FLIM images indicate chemical biomarkers including increased nicotinamide adenine dinucleotide and reduced collagen emission. While both sets of biomarkers can differentiate normal and cancerous tissue, we believe their combination will enable the discrimination of benign lesions possessing some of the indicated biomarkers, e.g., scarring or inflammation.

Automated classification of optical coherence tomography images for the diagnosis of oral malignancy in the hamster cheek pouch.

Most studies evaluating the potential of optical coherence tomography (OCT) for the diagnosis of oral cancer are based on visual assessment of OCT B-scans by trained experts. Human interpretation of the large pool of data acquired by modern high-speed OCT systems, however, can be cumbersome and extremely time consuming. Development of image analysis methods for automated and quantitative OCT image analysis could therefore facilitate the evaluation of such a large volume of data. We report automated algorithms for quantifying structural features that are associated with the malignant transformation of the oral epithelium based on image processing of OCT data. The features extracted from the OCT images were used to design a statistical classification model to perform the automated tissue diagnosis. The sensitivity and specificity of distinguishing malignant lesions from benign lesions were found to be 90.2% and 76.3%, respectively. The results of the study demonstrate the feasibility of using quantitative image analysis algorithms for extracting morphological features from OCT images to perform the automated diagnosis of oral malignancies in a hamster cheek pouch model.

Miniature, minimally invasive, tunable endoscope for investigation of the middle ear.

We demonstrate a miniature, tunable, minimally invasive endoscope for diagnosis of the auditory system. The probe is designed to sharply image anatomical details of the middle ear without the need for physically adjusting the position of the distal end of the endoscope. This is achieved through the addition of an electrowetted, tunable, electronically-controlled lens to the optical train. Morphological imaging is enabled by scanning light emanating from an optical coherence tomography system. System performance was demonstrated by imaging part of the ossicular chain and wall of the middle ear cavity of a normal mouse. During the experiment, we electronically moved the plane of best focus from the incudo-stapedial joint to the stapedial artery. Repositioning the object plane allowed us to image anatomical details of the middle ear beyond the depth of field of a static optical system. We also demonstrated for the first time to our best knowledge, that an optical system with an electrowetted, tunable lens may be successfully employed to measure sound-induced vibrations within the auditory system by measuring the vibratory amplitude of the tympanic membrane in a normal mouse in response to pure tone stimuli.

Simultaneous morphological and biochemical endogenous optical imaging of atherosclerosis

AIMS The aim of this study was to validate novel imaging technology for simultaneous morphological and biochemical endogenous optical imaging of coronary atherosclerotic plaque. METHODS AND RESULTS Optical coherence tomography (OCT) generates high-resolution 3D images of plaque morphology and endogenous fluorescence lifetime imaging microscopy (FLIM) characterizes biochemical composition. Both imaging modalities rely on plaques intrinsic optical characteristics, making contrast agents unnecessary. A multimodal OCT/FLIM system was utilized to generate luminal biochemical maps superimposed on high-resolution (7 µm axial and 13 µm lateral) structural volumetric images. Forty-seven fresh postmortem human coronary segments were imaged: pathological intimal thickening (PIT, n = 26), fibroatheroma (FA, n = 12), thin-cap FA (TCFA, n = 2), and fibrocalcific plaque (CA, n = 7), determined by histopathology. Multimodal images were evaluated, and each plaque identified as PIT, FA, TCFA, or CA based on expert OCT readers, and as having high-lipid (HL), high-collagen (HC), or low-collagen/low-lipid (LCL) luminal composition based on linear discriminant analysis of FLIM. Of 47 plaques, 89.4% (42/47) of the plaques were correctly identified based on OCT/FLIM evaluation using tissue histopathology and immunohistochemistry as the gold standard. Four of the misclassifications corresponded to confusing PIT with HL luminal composition for FA with HL cap. The other corresponded to confusing FA with a HC cap for FA with an LCL cap. CONCLUSION We have demonstrated the feasibility of accurate simultaneous OCT/FLIM morphological and biochemical characterization of coronary plaques at spatial resolutions and acquisition speeds compatible with catheter-based intravascular imaging. The success of this pilot study sets up future development of a multimodal intravascular imaging system that will enable studies that could help improve our understanding of plaque pathogenesis.

Multimodal optical coherence tomography and fluorescence lifetime imaging with interleaved excitation sources for simultaneous endogenous and exogenous fluorescence

Multimodal imaging probes a variety of tissue properties in a single image acquisition by merging complimentary imaging technologies. Exploiting synergies amongst the data, algorithms can be developed that lead to better tissue characterization than could be accomplished by the constituent imaging modalities taken alone. The combination of optical coherence tomography (OCT) with fluorescence lifetime imaging microscopy (FLIM) provides access to detailed tissue morphology and local biochemistry. The optical system described here merges 1310 nm swept-source OCT with time-domain FLIM having excitation at 355 and 532 nm. The pulses from 355 and 532 nm lasers have been interleaved to enable simultaneous acquisition of endogenous and exogenous fluorescence signals, respectively. The multimodal imaging system was validated using tissue phantoms. Nonspecific tagging with Alexa Flour 532 in a Watanbe rabbit aorta and active tagging of the LOX-1 receptor in human coronary artery, demonstrate the capacity of the system for simultaneous acquisition of OCT, endogenous FLIM, and exogenous FLIM in tissues.

Automated analysis of multimodal fluorescence lifetime imaging and optical coherence tomography data for the diagnosis of oral cancer in the hamster cheek pouch model

It is known that the progression of oral cancer is accompanied by changes in both tissue biochemistry and morphology. A multimodal imaging approach combining functional and structural imaging modalities could therefore provide a more comprehensive prognosis of oral cancer. This idea forms the central theme of the current study, wherein this premise is examined in the context of a multimodal imaging system that combines fluorescence lifetime imaging (FLIM) and optical coherence tomography (OCT). Towards this end, in the first part of the present study, the diagnostic advantage obtained by using both fluorescence intensity and lifetime information is assessed. In the second part of the study, the diagnostic potential of FLIM-derived biochemical features is compared with that of OCT-derived morphological features. For an objective assessment, several quantitative biochemical and morphological features from FLIM and OCT data, respectively, were obtained using signal and image processing techniques. These features were subsequently used in a statistical classification framework to quantify the diagnostic potential of different features. The classification accuracy for combined FLIM and OCT features was estimated to be 87.4%, which was statistically higher than accuracy based on only FLIM (83.2%) or OCT (81.0%) features. Moreover, the complimentary information provided by FLIM and OCT features, resulted in highest sensitivity and specificity for the combined FLIM and OCT features for discriminating benign (88.2% sens., 92.0% spec.), pre-cancerous (81.5% sens., 96.0% spec.), and cancerous (90.1% sens., 92.0% spec.) classes.

Multimodal optical imaging for simultaneous in-vivo morphological and biochemical characterization of oral epithelial cancer

Early detection of cancer is key to reducing morbidity and mortality. Morphological and chemical biomarkers presage the transition from normal to cancerous tissue. We have developed a noninvasive imaging system incorporating optical coherence tomography and fluorescence lifetime imaging to acquire both sets of biomarkers. Here we report early favorable results from an animal study designed to measure the capacity of this approach for early diagnosis of oral cancer.

Multimodality optical imaging combining optical coherence tomography (OCT) and fluorescence lifetime imaging (FLIM) for morphological and biochemical tissue characterization

Here we report on our current efforts to simultaneously quantify both morphological and biochemical tissue information by combining optical coherence tomography (OCT) and fluorescence lifetime imaging (FLIM). The Fourier domain OCT module is built around a custom designed high-speed spectrometer (bandwidth of 102 nm, 3 dB rolloff of 1.2 mm, lines rates of up to 59 kHz). A 40 nm bandwidth SLED centered at 830 nm provided an axial resolution of 7.6 mm for OCT. The objective lens provided 10 um lateral resolution for OCT and 100 um for FLIM. Lateral OCT and FLIM beam scanning was accomplished using a set of galvo mirrors. The FLIM module excites and collects the fluorescence decay signal pixel by pixel coincident with OCT A-line collection. Each 2-D FLIM image has a corresponding coregistered OCT volume. Fluorescence excitation for FLIM was provided by a solid-state pulse laser (355 nm, 1 ns FWHM, 50 kHz rep rate). The fluorescence signal was detected with a MCP-PMT coupled to a 1.5 GHz digitizer (250 ps temporal resolutions). In addition, simultaneous multispectral time-resolved fluorescence detection was achieved by separating the fluorescence emission in three bands using a series of dichroic mirrors and bandpass filters, and launching each band into three fibers of different lengths (providing a time delay of 50 ns among bands) focused onto the MCP-PMT. The resulting OCT/FLIM system is capable of a maximum A-line rate of 59 kHz for OCT and a maximum pixel rate of at least 30 kHz for FLIM. The multimodality OCT/FLIM imaging system was validated on biological tissue. Future efforts include evaluating its potential for oral cancer diagnosis and intravascular imaging.