Showing papers by "Stephen A. Boppart published in 2016"

Stain-free histopathology by programmable supercontinuum pulses

Abstract: The preparation, staining, visualization and interpretation of histological images of tissue is well accepted as the gold standard process for the diagnosis of disease. These methods have a long history of development, and are used ubiquitously in pathology, despite being highly time- and labour-intensive. Here, we introduce a unique optical imaging platform and methodology for label-free multimodal multiphoton microscopy that uses a novel photonic-crystal fibre source to generate tailored chemical contrast based on programmable supercontinuum pulses. We demonstrate the collection of optical signatures of the tumour microenvironment, including evidence of mesoscopic biological organization, tumour cell migration and (lymph-) angiogenesis collected directly from fresh ex vivo mammary tissue. Acquisition of these optical signatures and other cellular or extracellular features, which are largely absent from histologically processed and stained tissue, combined with an adaptable platform for optical alignment-free programmable-contrast imaging, offers the potential to translate stain-free molecular histopathology into routine clinical use. A single-source multimodal nonlinear optical imaging system has been developed to probe different endogenous biomolecules. Rapid, stain-free imaging of fresh tissue specimens is possible with short turnaround times for disease diagnosis.

Response of Simulated Drinking Water Biofilm Mechanical and Structural Properties to Long-Term Disinfectant Exposure

Abstract: Mechanical and structural properties of biofilms influence the accumulation and release of pathogens in drinking water distribution systems (DWDS). Thus, understanding how long-term residual disinfectants exposure affects biofilm mechanical and structural properties is a necessary aspect for pathogen risk assessment and control. In this study, elastic modulus and structure of groundwater biofilms was monitored by atomic force microscopy (AFM) and optical coherence tomography (OCT) during three months of exposure to monochloramine or free chlorine. After the first month of disinfectant exposure, the mean stiffness of monochloramine- or free-chlorine-treated biofilms was 4 to 9 times higher than those before treatment. Meanwhile, the biofilm thickness decreased from 120 ± 8 μm to 93 ± 6-107 ± 11 μm. The increased surface stiffness and decreased biofilm thickness within the first month of disinfectant exposure was presumably due to the consumption of biomass. However, by the second to third month during disinfectant exposure, the biofilm mean stiffness showed a 2- to 4-fold decrease, and the biofilm thickness increased to 110 ± 7-129 ± 8 μm, suggesting that the biofilms adapted to disinfectant exposure. After three months of the disinfectant exposure process, the disinfected biofilms showed 2-5 times higher mean stiffness (as determined by AFM) and 6-13-fold higher ratios of protein over polysaccharide, as determined by differential staining and confocal laser scanning microscopy (CLSM), than the nondisinfected groundwater biofilms. However, the disinfected biofilms and nondisinfected biofilms showed statistically similar thicknesses (t test, p > 0.05), suggesting that long-term disinfection may not significantly remove net biomass. This study showed how biofilm mechanical and structural properties vary in response to a complex DWDS environment, which will contribute to further research on the risk assessment and control of biofilm-associated-pathogens in DWDS.

Intraoperative optical coherence tomography for assessing human lymph nodes for metastatic cancer

Abstract: Evaluation of lymph node (LN) status is an important factor for detecting metastasis and thereby staging breast cancer. Currently utilized clinical techniques involve the surgical disruption and resection of lymphatic structure, whether nodes or axillary contents, for histological examination. While reasonably effective at detection of macrometastasis, the majority of the resected lymph nodes are histologically negative. Improvements need to be made to better detect micrometastasis, minimize or eliminate lymphatic disruption complications, and provide immediate and accurate intraoperative feedback for in vivo cancer staging to better guide surgery. We evaluated the use of optical coherence tomography (OCT), a high-resolution, real-time, label-free imaging modality for the intraoperative assessment of human LNs for metastatic disease in patients with breast cancer. We assessed the sensitivity and specificity of double-blinded trained readers who analyzed intraoperative OCT LN images for presence of metastatic disease, using co-registered post-operative histopathology as the gold standard. Our results suggest that intraoperative OCT examination of LNs is an appropriate real-time, label-free, non-destructive alternative to frozen-section analysis, potentially offering faster interpretation and results to empower superior intraoperative decision-making. Intraoperative OCT has strong potential to supplement current post-operative histopathology with real-time in situ assessment of LNs to preserve both non-cancerous nodes and their lymphatic vessels, and thus reduce the associated risks and complications from surgical disruption of lymphoid structures following biopsy.

Raman Spectroscopic Analysis Reveals Abnormal Fatty Acid Composition in Tumor Micro- and Macroenvironments in Human Breast and Rat Mammary Cancer.

Abstract: Fatty acids play essential roles in the growth and metastasis of cancer cells. To facilitate their avid growth and proliferation, cancer cells not only alter the fatty acid synthesis and metabolism intracellularly and extracellularly, but also in the macroenvironment via direct or indirect pathways. We report here, using Raman micro-spectroscopy, that an increase in the production of polyunsaturated fatty acids (PUFAs) was identified in both cancerous and normal appearing breast tissue obtained from breast cancer patients and tumor-bearing rats. By minimizing confounding effects from mixed chemicals and optimizing the signal-to-noise ratio of Raman spectra, we observed a large-scale transition from monounsaturated fatty acids to PUFAs in the tumor while only a small subset of fatty acids transitioned to PUFAs in the tumor micro- and macroenvironment. These data have important implications for further clarifying the macroenvironmental effect of cancer progression and provide new potential approaches for characterizing the tumor micro- and macroenvironment of breast cancer in both pre-clinical animal studies and clinical applications.

Progress in Cherenkov femtosecond fiber lasers

Abstract: We review the recent developments in the field of ultrafast Cherenkov fiber lasers. Two essential properties of such laser systems - broad wavelength tunability and high efficiency of Cherenkov radiation wavelength conversion are discussed. The exceptional performance of the Cherenkov fiber laser systems are highlighted - dependent on the realization scheme, the Cherenkov lasers can generate the femtosecond output tunable across the entire visible and even the UV range, and for certain designs more than 40 % conversion efficiency from the pump to Cherenkov signal can be achieved. The femtosecond Cherenkov laser with all-fiber architecture is presented and discussed. Operating in the visible range, it delivers 100-200 fs wavelength-tunable pulses with multimilliwatt output power and exceptionally low noise figure an order of magnitude lower than the traditional wavelength tunable supercontinuum-based femtosecond sources. The applications for Cherenkov laser systems in practical biophotonics and biomedical applications, such as bio-imaging and microscopy, are discussed.

A Mosaicking Approach for In Vivo Thickness Mapping of the Human Tympanic Membrane Using Low Coherence Interferometry

Abstract: The thickness of the human tympanic membrane (TM) is known to vary considerably across different regions of the TM. Quantitative determination of the thickness distribution and mapping of the TM is of significant importance in hearing research, particularly in mathematical modeling of middle-ear dynamics. Change in TM thickness is also associated with several middle-ear pathologies. Determination of the TM thickness distribution could therefore also enable a more comprehensive diagnosis of various otologic diseases. Despite its importance, very limited data on human TM thickness distribution, obtained almost exclusively from ex vivo samples, are available in the literature. In this study, the thickness distribution for the in vivo human TM is reported for the first time. A hand-held imaging system, which combines a low coherence interferometry (LCI) technique for single-point thickness measurement, with video-otoscopy for recording the image of the TM, was used to collect the data used in this study. Data were acquired by pointing the imaging probe over different regions of the TM, while simultaneously recording the LCI and concomitant TM surface video image data from an average of 500 locations on the TM. TM thickness distribution maps were obtained by mapping the LCI imaging sites onto an anatomically accurate wide-field image of the TM, which was generated by mosaicking the sequence of multiple small field-of-view video-otoscopy images. Descriptive statistics of the thickness measurements obtained from the different regions of the TM are presented, and the general thickness distribution trends are discussed.

Automated computational aberration correction method for broadband interferometric imaging techniques.

Abstract: Numerical correction of optical aberrations provides an inexpensive and simpler alternative to the traditionally used hardware-based adaptive optics techniques. In this Letter, we present an automated computational aberration correction method for broadband interferometric imaging techniques. In the proposed method, the process of aberration correction is modeled as a filtering operation on the aberrant image using a phase filter in the Fourier domain. The phase filter is expressed as a linear combination of Zernike polynomials with unknown coefficients, which are estimated through an iterative optimization scheme based on maximizing an image sharpness metric. The method is validated on both simulated data and experimental data obtained from a tissue phantom, an ex vivo tissue sample, and an in vivo photoreceptor layer of the human retina.

Detection of retinal blood vessel changes in multiple sclerosis with optical coherence tomography.

Abstract: Although retinal vasculitis is common in multiple sclerosis (MS), it is not known if MS is associated with quantitative abnormalities in retinal blood vessels (BVs). Optical coherence tomography (OCT) is suitable for examining the integrity of the anterior visual pathways in MS. In this paper we have compared the size and number of retinal blood vessels in patients with MS, with and without a history of optic neuritis (ON), and control subjects from the cross-sectional retinal images from OCT. Blood vessel diameter (BVD), blood vessel number (BVN), and retinal nerve fiber layer thickness (RNFLT) were extracted from OCT images collected from around the optic nerves of 129 eyes (24 control, 24 MS + ON, 81 MS-ON) of 71 subjects. Associations between blood vessel metrics, MS diagnosis, MS disability, ON, and RNFLT were evaluated using generalized estimating equation (GEE) models. MS eyes had a lower total BVD and BVN than control eyes. The effect was more pronounced with increased MS disability, and persisted in multivariate models adjusting for RNFLT and ON history. Twenty-nine percent (29%) of MS subjects had fewer retinal blood vessels than all control subjects. MS diagnosis, disability, and ON history were not associated with average blood vessel size. The relationship between MS and lower total BVD/BVN is not accounted for by RNFLT or ON. Further study is needed to determine the relationship between OCT blood vessel metrics and qualitative retinal blood vessel abnormalities in MS.

Sensor-Based Technique for Manually Scanned Hand-Held Optical Coherence Tomography Imaging.

Abstract: Hand-held optical coherence tomography (OCT) imaging probes offer flexibility to image sites that are otherwise challenging to access. While the majority of hand-held imaging probes utilize galvanometer- or MEMS-scanning mirrors to transversely scan the imaging beam, these probes are commonly limited to lateral fields-of-view (FOV) of only a few millimeters. The use of a freehand manually scanned probe can significantly increase the lateral FOV. However, using the traditional fixed-rate triggering scheme for data acquisition in a manually scanned probe results in imaging artifacts due to variations in the scan velocity of the imaging probe. These artifacts result in a structurally inaccurate image of the sample. In this paper, we present a sensor-based manual scanning technique for OCT imaging, where real-time feedback from an optical motion sensor is used to trigger data acquisition. This technique is able to circumvent the problem of motion artifacts during manual scanning by adaptively altering the trigger rate based on the instantaneous scan velocity, enabling OCT imaging over a large lateral FOV. The feasibility of the proposed technique is demonstrated by imaging several biological and nonbiological samples.

Magnetomotive Optical Coherence Elastography for Magnetic Hyperthermia Dosimetry Based on Dynamic Tissue Biomechanics

Abstract: Magnetic nanoparticles (MNPs) have been used in many diagnostic and therapeutic biomedical applications over the past few decades to enhance imaging contrast, steer drugs to targets, and treat tumors via hyperthermia. Optical coherence tomography (OCT) is an optical biomedical imaging modality that relies on the detection of backscattered light to generate high-resolution cross-sectional images of biological tissue. MNPs have been utilized as imaging contrast and perturbative mechanical agents in OCT in techniques called magnetomotive OCT (MM-OCT) and magnetomotive elastography (MM-OCE), respectively. MNPs have also been independently used for magnetic hyperthermia treatments, enabling therapeutic functions such as killing tumor cells. It is well known that the localized tissue heating during hyperthermia treatments result in a change in the biomechanical properties of the tissue. Therefore, we propose a novel dosimetric technique for hyperthermia treatment based on the viscoelasticity change detected by MM-OCE, further enabling the theranostic function of MNPs. In this paper, we first review the basic principles and applications of MM-OCT, MM-OCE, and magnetic hyperthermia, and present new preliminary results supporting the concept of MM-OCE-based hyperthermia dosimetry.

Automated interferometric synthetic aperture microscopy and computational adaptive optics for improved optical coherence tomography

Abstract: In this paper, we introduce an algorithm framework for the automation of interferometric synthetic aperture microscopy (ISAM). Under this framework, common processing steps such as dispersion correction, Fourier domain resampling, and computational adaptive optics aberration correction are carried out as metrics-assisted parameter search problems. We further present the results of this algorithm applied to phantom and biological tissue samples and compare with manually adjusted results. With the automated algorithm, near-optimal ISAM reconstruction can be achieved without manual adjustment. At the same time, the technical barrier for the nonexpert using ISAM imaging is also significantly lowered.

Intravascular magnetomotive optical coherence tomography of targeted early‐stage atherosclerotic changes in ex vivo hyperlipidemic rabbit aortas

Abstract: Representative magnetomotive signal (green) using targeted and non-targeted magnetomotive microspheres in atherosclerotic diseased rabbit aortas. We report the development of an intravascular magnetomotive optical coherence tomography (IV-MM-OCT) system used with targeted protein microspheres to detect early-stage atherosclerotic fatty streaks/plaques. Magnetic microspheres (MSs) were injected in vivo in rabbits, and after 30 minutes of in vivo circulation, excised ex vivo rabbit aorta samples specimens were then imaged ex vivo with our prototype IV-MM-OCT system. The alternating magnetic field gradient was provided by a unique pair of external custom-built electromagnetic coils that modulated the targeted magnetic MSs. The results showed a statistically significant MM-OCT signal from the aorta samples specimens injected with targeted MSs.

Rapid diagnosis and differentiation of microbial pathogens in otitis media with a combined Raman spectroscopy and low-coherence interferometry probe: toward in vivo implementation.

Abstract: We investigate and demonstrate the feasibility of using a combined Raman scattering (RS) spectroscopy and low-coherence interferometry (LCI) probe to differentiate microbial pathogens and improve our diagnostic ability of ear infections [otitis media (OM)]. While the RS probe provides noninvasive molecular information to identify and differentiate infectious microorganisms, the LCI probe helps to identify depth-resolved structural information as well as to guide and monitor positioning of the Raman spectroscopy beam for relatively longer signal acquisition times. A series of phantom studies, including the use of human middle ear effusion samples, were performed to mimic the conditions of in vivo investigations. These were also conducted to validate the feasibility of using this combined RS/LCI probe for point-of-care diagnosis of the infectious pathogen(s) in OM patients. This work establishes important parameters for future in vivo investigations of fast and accurate determination and diagnosis of infectious microorganisms in OM patients, potentially improving the efficacy and outcome of OM treatments, and importantly reducing the misuse of antibiotics in the presence of viral infections.

Longitudinal in vivo tracking of adverse effects following topical steroid treatment

Abstract: Topical steroids are known for their anti-inflammatory properties and are commonly prescribed to treat many adverse skin conditions such as eczema and psoriasis. While these treatments are known to be effective, adverse effects including skin atrophy are common. In this study, the progression of these effects is investigated in an in vivo mouse model using multimodal optical microscopy. Utilizing a system capable of performing two-photon excitation fluorescence microscopy (TPEF) of reduced nicotinamide adenine dinucleotide (NADH) to visualize the epidermal cell layers and second harmonic generation (SHG) microscopy to identify collagen in the dermis, these processes can be studied at the cellular level. Fluorescence lifetime imaging microscopy (FLIM) is also utilized to image intracellular NADH levels to obtain molecular information regarding metabolic activity following steroid treatment. In this study, fluticasone propionate (FP)-treated, mometasone furoate (MF)-treated and untreated animals were imaged longitudinally using a custom-built multimodal optical microscope. Prolonged steroid treatment over the course of 21 days is shown to result in a significant increase in mean fluorescence lifetime of NADH, suggesting a faster rate of maturation of epidermal keratinocytes. Alterations to collagen organization and the structural microenvironment are also observed. These results give insight into the structural and biochemical processes of skin atrophy associated with prolonged steroid treatment.

Computed Optical Interferometric Imaging: Methods, Achievements, and Challenges

Abstract: Three-dimensional high-resolution optical imaging systems are generally restricted by the tradeoff between resolution and depth-of-field as well as imperfections in the imaging system or sample. Computed optical interferometric imaging is able to overcome these longstanding limitations using methods such as interferometric synthetic aperture microscopy (ISAM) and computational adaptive optics (CAO), which manipulate the complex interferometric data. These techniques correct for limited depth-of-field and optical aberrations without the need for additional hardware. This paper aims to outline these computational methods, making them readily available to the research community. Achievements of the techniques will be highlighted, along with past and present challenges in implementing the techniques. Challenges such as phase instability and determination of the appropriate aberration correction have been largely overcome so that imaging of living tissues using ISAM and CAO is now possible. Computed imaging in optics is becoming a mature technology poised to make a significant impact in medicine and biology.

In vivo evaluation of adipose- and muscle-derived stem cells as a treatment for nonhealing diabetic wounds using multimodal microscopy

Abstract: Impaired skin wound healing is a significant comorbid condition of diabetes, which often results in nonhealing diabetic ulcers due to poor peripheral microcirculation, among other factors. The effectiveness of the regeneration of adipose-derived stem cells (ADSCs) and muscle-derived stem cells (MDSCs) was assessed using an integrated multimodal microscopy system equipped with two-photon fluorescence and second-harmonic generation imaging. These imaging modalities, integrated in a single platform for spatial and temporal coregistration, allowed us to monitor in vivo changes in the collagen network and cell dynamics in a skin wound. Fluorescently labeled ADSCs and MDSCs were applied topically to the wound bed of wild-type and diabetic (db/db) mice following punch biopsy. Longitudinal imaging demonstrated that ADSCs and MDSCs provided remarkable capacity for improved diabetic wound healing, and integrated microscopy revealed a more organized collagen remodeling in the wound bed of treated mice. The results from this study verify the regenerative capacity of stem cells toward healing and, with multimodal microscopy, provide insight regarding their impact on the skin microenvironment. The optical method outlined in this study, which has the potential for in vivo human use, may optimize the care and treatment of diabetic nonhealing wounds.

- Optical Coherence Tomography: Overview and Applications

Abstract: Owing to these unique features, OCT research advances have been gaining increasing attention and are nding numerous applications in areas of biological research and medical practice. In the early 1990s, when the technology was rst demonstrated, OCT quickly found a niche in ophthalmology, where it provided the rst in situ high-resolution 3D images of structures in the eye (Drexler and Fujimoto 2008b), including the retina, cornea, and the optic disc. Before long, the advantages of OCT were recognized in many other elds, including cell biology (Boppart et al. 1998; Drexler et al. 1999), dermatology (Gambichler et al. 2005; Weissman et al. 2004), cancer research (Zhou et al. 2010; Zysk et al. 2007), dentistry (Colston et al. 1998; Otis et al. 2000), and tissue engineering (Liang et al. 2009; Matcher 2011), to name a few. e invention and development of endoscopy and catheter OCT devices (Herz et al. 2004; Tearney et al. 1996, 1997) has extended the use of this technology to cardiovascular (Boppart et al. 1997; Jenkins et al. 2006), gastrointestinal (Herz et al. 2004; Rollins et al. 1999), urological (Wang et al. 2011), and gynecological (Jackle et al. 2000) applications. Today, OCT continues to develop rapidly in many areas, with new technologies and variant modalities emerging, which are not only improving the performance of OCT in terms of resolution, contrast, functional analysis capability, and imaging speed but also bringing it to new elds of application. In this chapter, we will introduce the working principle of OCT, review recent advances in OCT technology and applications, and exploit the unique features of OCT for cellular imaging, particularly for the investigation of cell activity and dynamics in in vivo skin and engineered tissues.

Filtering for unwrapping noisy Doppler optical coherence tomography images for extended microscopic fluid velocity measurement range.

Abstract: In this Letter, we report the first application of two phase denoising algorithms to Doppler optical coherence tomography (DOCT) velocity maps. When combined with unwrapping algorithms, significantly extended fluid velocity dynamic range is achieved. Instead of the physical upper bound, the fluid velocity dynamic range is now limited by noise level. We show comparisons between physical simulated ideal velocity maps and the experimental results of both algorithms. We demonstrate unwrapped DOCT velocity maps having a peak velocity nearly 10 times the theoretical measurement range.

Computational adaptive optics of the human retina

Abstract: It is well known that patient-specific ocular aberrations limit imaging resolution in the human retina. Previously, hardware adaptive optics (HAO) has been employed to measure and correct these aberrations to acquire high-resolution images of various retinal structures. While the resulting aberration-corrected images are of great clinical importance, clinical use of HAO has not been widespread due to the cost and complexity of these systems. We present a technique termed computational adaptive optics (CAO) for aberration correction in the living human retina without the use of hardware adaptive optics components. In CAO, complex interferometric data acquired using optical coherence tomography (OCT) is manipulated in post-processing to adjust the phase of the optical wavefront. In this way, the aberrated wavefront can be corrected. We summarize recent results in this technology for retinal imaging, including aberration-corrected imaging in multiple retinal layers and practical considerations such as phase stability and image optimization.

Ratiometric analysis of in vivo retinal layer thicknesses in multiple sclerosis.

Abstract: We performed ratiometric analysis of retinal optical coherence tomography images for the first time in multiple sclerosis (MS) patients. The ratiometric analysis identified differences in several retinal layer thickness ratios in the cohort of MS subjects without a history of optic neuritis (ON) compared to healthy control (HC) subjects, and there was no difference in standard retinal nerve fiber layer thickness (RNFLT). The difference in such ratios between HC subjects and those with mild MS-disability, without a difference in RNFLT, further suggests the possibility of using layer ratiometric analysis for detecting early retinal changes in MS. Ratiometric analysis may be useful and potentially more sensitive for detecting disease changes in MS.

Phase measurement, analysis, and correction methods for coherent imaging systems

Abstract: Methods and apparatus for assessing and correcting phase variations and motion artifacts in a coherent tomogram of a sample. Coherent techniques are used scan a broadband optical beam across a sample in a specified scan pattern and to acquire a cube of complex data constituting a full tomogram. Generalized motion of the sample is then quantified based at least on a phase variation measured during the course of scanning the broadband optical beam in the specified scan pattern. Generalized motion includes both actual motion and apparent motion due to organized variation of some physical parameter such as temperature. Intensity structure of speckle imaged during the course of coherently acquiring the full tomograpm may be used to correct for motion of the sample in a plane transverse to a depth axis along the incident beam.

Filtering and unwrapping doppler optical coherence tomography velocity maps

Abstract: In this abstract, we describe two computational methods for filtering and unwrapping Doppler optical coherence tomography velocity maps, which enable extended measurement range of fluid velocity. We further demonstrate experimental results of both methods and compare to physical simulations.

Correction of aberrations in the human eye using computational methods.

Abstract: Phase-sensitive imaging and computational correction of patient-specific optical aberrations enable high-resolution imaging of the human retina to aid diagnosis and treatment of eye diseases.

Intraoperative In Vivo Assessment of Lymph Nodes with Optical Coherence Tomography

Abstract: Techniques have been developed to localize sentinel lymph nodes during cancer surgery and for post-operative histology. Intraoperative OCT, however, uniquely offers microscopic label-free in vivo assessment of lymph nodes for metastatic disease.

Intraoperative OCT for soft tissue sarcoma margin identification

Abstract: Sarcomas are rare but highly aggressive tumors, and recurrence after surgical excision remains high. Tumor margin delineation in feline and canine patients was achieved with intraoperative OCT to help accomplish and validate complete resection.

Spectroscopic Raman Analysis of Tumor Micro- and Macroenvironments in Human Breast and Rat Mammary Cancer

Abstract: The tumor microenvironment plays important roles in carcinogenesis. We report spectroscopic Raman analysis of fatty acids and increased polyunsaturated fatty acids in the wider tumor macroenvironment in both human breast and rat mammary cancer.