In this letter, we report the first use of oblique incidence diffuse reflectance spectrometry to conduct in-vivo measurements of optical properties of three different types of pigmented skin lesions, including melanoma, dysplastic, and common nevi. Both absorption and reduced scattering coefficient spectra were estimated from the spatially resolved diffuse reflectance within the wavelength range of 455-765 nm for 144 pigmented skin lesions including 16 melanomas. The absorption and reduced scattering spectra were found to change with the malignancy of the skin lesions, which were generally higher for the malignant cases than the benign ones. Based on the measurement results, the physiological origin leading to the change of the absorption and scattering properties is also discussed.

A new approach to distinguish normal fat, well-differentiated (WD), and dedifferentiated liposarcoma (LS) tumors is demonstrated, based on the use of optical coherence tomography (OCT). OCT images show the same structures seen with conventional histological methods. Our visual grading analysis is supported by numerical analysis of observed structures for normal fat and WDLS samples. Further development could apply the real-time and high resolution advantages of OCT for use in liposarcoma diagnosis and clinical procedures.

Osteosarcoma is one of the most common primary malignant tumors of the bone and the second leading cause of cancer-related deaths in the pediatric age group. Confirmed diagnosis and prompt treatment of osteosarcoma are critical for effective prognosis. In this study, we investigate the application of photoacoustic imaging (PAI) for the detection of osteosarcoma in an animal model. Cross-section images of a normal rat leg and a tumorous rat leg were successfully reconstructed in vivo. Morphological changes and the development of the implanted osteosarcoma were accurately mapped with time-dependent photoacoustic images. Furthermore, we evaluate the use of gold nanorods as contrast agents for imaging osteosarcoma with PAI. This is the first study that uses PAI to detect osteosarcoma invivo, and the results suggest that PAI has the potential clinical application for detecting osteosarcoma in the early stage.

We propose a rate process model for describing photochemical damage to retinal cells by short wavelength laser exposures. The rate equation for photochemical damage contains a positive rate that is temperature independent, and a negative (quenching) rate that is temperature dependent. Using the traditional Arrhenius integral to describe thermal damage, we derive damage threshold doses for both thermal and photochemical mechanisms, and show that the model accounts for the sharp transition from thermal to photochemical damage thresholds that have recently been observed in an in-vitro retinal model.

In Fourier domain optical coherence tomography (FD-OCT), a large amount of interference data needs to be resampled from the wavelength domain to the wavenumber domain prior to Fourier transformation. We present an approach to optimize this data processing, using a graphics processing unit (GPU) and parallel processing algorithms. We demonstrate an increased processing and rendering rate over that previously reported by using GPU paged memory to render data in the GPU rather than copying back to the CPU. This avoids unnecessary and slow data transfer, enabling a processing and display rate of well over 524,000 A-scan/s for a single frame. To the best of our knowledge this is the fastest processing demonstrated to date and the first time that FD-OCT processing and rendering has been demonstrated entirely on a GPU.

We present a technique to reduce speckle in optical coherence tomography images of soft tissues. An average is formed over a set of B-scans that have been decorrelated by viscoelastic creep strain. The necessary correction for the deformation-induced spatial distortions between B-scans is achieved through geometrical co-registration using an affine transformation. Speckle reduction by up to a factor of 1.65 is shown in images of tissue-mimicking soft fibrin phantoms and excised human lymph node tissue with no observable loss of spatial resolution.

We synthesize drug-loaded poly (lactic-co-glycolic acid) (PLGA) microspheres for image-guided combinatory epigenetic therapy in MCF-10A human mammary epithelial cells. LY294002 and Nile Red are encapsulated in microspheres for sustained drug release and fluorescence microscopic imaging. Drug-loaded microspheres target MCF-10A cells through a three-step binding process involving biotinylated antibody, streptavidin, and biotinylated microspheres. LY294002 loaded microspheres and 5-Aza-2-deoxycytidine are applied to MCF-10A cells for combinatory PI3K/AKT inhibition and deoxyribonucleic acid (DNA) demethylation. Our study implies the technical potential of disease targeting and image-guided combinatory epigenetic therapy using drug-loaded multifunctional biodegradable PLGA microspheres.

This PDF file contains the editorial “Special Section Guest Editorial: Coherent Raman Imaging Techniques and Biomedical Applications” for JBO Vol. 16 Issue 02

A simple scheme for video-rate wide-field coherent anti-Stokes Raman scattering (CARS) microscopy is presented. The method is based on collinear nonphase-matching illumination. The mechanisms leading to CARS signal generation are investigated. We find that refraction-mediated phase-matching is the main effect. Video-rate wide-field CARS microscopy of polystyrene beads and CARS wide-field images of C. elegans embryos are shown, and the capabilities and the limitations of the scheme are discussed.

We demonstrate the simultaneous collection and separation of femtosecond-laser-based forward-collected coherent anti-Stokes Raman scattering (F-CARS) and two-photon-excitation-induced fluorescence lifetime images (FLIM) using time-correlated single photon counting (TCSPC). We achieve this in a nondescanned geometry using a single multimode fiber without significant loss of light, field of view, and most importantly, TCSPC timing fidelity. In addition to showing the ability to separate CARS images from FLIM images using time gating, we also demonstrate composite multimodal epicollected FLIM imaging with fiber-collected F-CARS imaging in live cells.

The lipid distribution in the mouse meibomian gland was examined with picosecond spectral anti-Stokes Raman scattering (CARS) imaging. Spectral CARS data sets were generated by imaging specific localized regions of the gland within tissue sections at consecutive Raman shifts in the CH₂ stretching vibrational range. Spectral differences between the location specific CARS spectra obtained in the lipid-rich regions of the acinus and the central duct were observed, which were confirmed with a Raman microspectroscopic analysis, and attributed to meibum lipid modifications within the gland. A principal component analysis of the spectral data set reveals changes in the CARS spectrum when transitioning from the acini to the central duct. These results demonstrate the utility of picosecond spectral CARS imaging combined with multivariate analysis for assessing differences in the distribution and composition of lipids in tissues.

Multiplex coherent anti-Stokes Raman scattering (MCARS) provides labeling free and fast characterization of materials and biological samples in nonlinear microscopy. In spite of its success, remaining challenges regarding the data analysis for chemoselective imaging still have to be solved. In general, image contrast has been realized by using only one spectral feature directly taken from the unprocessed raw data. This procedure is limited to strong and well separated Raman resonances like the saturated CH-stretching vibration of lipids in the case of biological samples. In order to overcome this limitation, we present a new method of MCARS data processing that exploits the whole measured spectrum to disentangle overlapping contributions of different (bio-) chemical components. Our "two-step" approach is based on the combination of imaginary part extraction followed by global fitting of the hyperspectral data set. Previous knowledge about the sample, e.g., pure spectra of the individual components is no longer necessary. The result is a highly contrasted image, where the patterns and differences between the sample components can be represented in different colors. We successfully applied this method to complex structured polymer samples and biological tissues.

We present the assets and constraints of using optical parametric oscillators (OPOs) to perform point scanning nonlinear microscopy and spectroscopy with special emphasis on coherent Raman spectroscopy. The difterent possible configurations starting with one OPO and two OPOs are described in detail and with comments that are intended to be practically useful for the user. Explicit examples on test samples such as nonlinear organic crystal, polystyrene beads, and fresh mouse tissues are given. Special emphasis is given to background-free coherent Raman anti-Stokes scattering (CARS) imaging, including CARS hyperspectral imaging in a fully automated mode with commercial OPOs.

We experimentally compare the performance and image contrast of spontaneous Raman and coherent Stokes Raman scattering microscopy. We demonstrate the differences between these techniques on a series of geometry-controlled samples that range in complexity from a point (array of tips) to one-dimensional (line grating) and, lastly, two-dimensional (checkereboard) microstructure. Through the use of this sample series, a comparison of the focal volume, achievable signal-to-noise, and resulting image contrast is made. The results demonstrate the effciency and spatial resolution attainable in coherent Raman microscopy relative to spontaneous Raman microscopy. Additionally, we detail potential complications in the interpretation of coherent Raman images of sample fine structure, where contrast is no longer based solely on oscillator concentration but can be influenced by sample microstructure.

We perform multiplex coherent anti-Stokes Raman scattering (CARS) micro-spectroscopy with a picosecond pulsed laser and a broadband supercontinuum (SC) generated in photonic crystal fiber. CARS signal stability is achieved using an active fiber coupler that avoids thermal and mechanical drifts. We obtain multiplex CARS spectra for test liquids in the 600-2000 cm⁻¹ spectral range. In addition we investigate the polarization dependence of the CARS spectra when rotating the pump beam linear polarization state relative to the linearly polarized broad stokes SC. From these polarization measurements we deduce the Raman depolarization ratio, the resonant versus nonresonant contribution, the Raman resonance frequency, and the linewidth.

Myelin loss and axonal degeneration predominate in many neurological disorders; however, methods to visualize them simultaneously in live tissue are unavailable. We describe a new imaging strategy combining video rate reflectance and fluorescence confocal imaging with coherent anti-Stokes Raman scattering (CARS) microscopy tuned to CH₂ vibration of myelin lipids, applied in live tissue of animals with chronic experimental autoimmune encephalomyelitis (EAE). Our method allows monitoring over time of demyelination and neurodegeneration in brain slices with high spatial resolution and signal-to-noise ratio. Local areas of severe loss of lipid signal indicative of demyelination and loss of the reflectance signal from axons were seen in the corpus callosum and spinal cord of EAE animals. Even in myelinated areas of EAE mice, the intensity of myelin lipid signals is significantly reduced. Using heterozygous knock-in mice in which green fluorescent protein replaces the CX₃CR1 coding sequence that labels central nervous system microglia, we find areas of activated microglia colocalized with areas of altered reflectance and CARS signals reflecting axonal injury and demyelination. Our data demonstrate the use of multimodal CARS microscopy for characterization of demyelinating and neurodegenerative pathology in a mouse model of multiple sclerosis, and further confirm the critical role of microglia in chronic inflammatory neurodegeneration.

We demonstrate for the first time the applicability of multimodal nonlinear optical (NLO) microscopy to the interrogation of stented coronary arteries under different diet and stent deployment conditions. Bare metal stents and Taxus drug-eluting stents (DES) were placed in coronary arteries of Ossabaw pigs of control and atherogenic diet groups. Multimodal NLO imaging was performed to inspect changes in arterial structures and compositions after stenting. Sum frequency generation, one of the multimodalities, was used for the quantitative analysis of collagen content in the peristent and in-stent artery segments of both pig groups. Atherogenic diet increased lipid and collagen in peristent segments. In-stent segments showed decreased collagen expression in neointima compared to media. Deployment of DES in atheromatous arteries inhibited collagen expression in the arterial media.

We demonstrate the real-time imaging of laser-induced disruption of the cellular membrane in a living HeLa cell and its cellular response with a multifocus coherent anti-Stokes Raman scattering (CARS) microscope. A near-infrared pulsed laser beam tightly focused on the cellular membrane of a living cell induces ablation at the focal point causing a local disruption of the cellular membrane. After the membrane disruption a dark spot decreasing CARS intensity of 2840 cm−¹ Raman shift at the disrupted site appears. This dark spot immediately disappears and a strong CARS signal is observed around the disrupted site. This increase of the CARS signal might be caused by resealing of the disrupted site via aggregation of the patch lipid vesicles in the cytoplasm. The accumulation of lipids around the disrupted site is also confirmed with three-dimensional CARS images of a cell before and after membrane disruption. The temporal behavior of the CARS signal at the disrupted site is observed to detect the fusion dynamics of patch vesicles

We demonstrate two different coherent anti-Stokes Raman scattering (CARS) microscopy and microspectroscopy methods based on the spectral focusing mechanism. The first method uses strongly chirped broadband pulses from a single Ti:sapphire laser and generates CARS signals at the fingerprint region. Fast modulation of the time delay between the pump and Stokes laser pulses coupled with lock-in signal detection significantly reduces the nonresonant background and produces Raman-like CARS signals with a spectral resolution of 20 cm−¹. The second method generates CARS signals in the CH (carbon-hydrogen) stretching region with IR supercontinuum pulses from a photonic crystal fiber. The spectral resolution of 30 cm−¹ is achieved. Maximum entropy method is used to retrieve a Raman-equivalent CARS spectrum from lipid membranes. Chemical imaging and microspectroscopy are demonstrated with various samples.

Contemporary brain tumor research focuses on two challenges: First, tumor typing and grading by analyzing excised tissue is of utmost importance for choosing a therapy. Second, for prognostication the tumor has to be removed as completely as possible. Nowadays, histopathology of excised tissue using haematoxylin-eosine staining is the gold standard for the definitive diagnosis of surgical pathology specimens. However, it is neither applicable in vivo, nor does it allow for precise tumor typing in those cases when only nonrepresentative specimens are procured. Infrared and Raman spectroscopy allow for very precise cancer analysis due to their molecular specificity, while nonlinear microscopy is a suitable tool for rapid imaging of large tissue sections. Here, unstained samples from the brain of a domestic pig have been investigated by a multimodal nonlinear imaging approach combining coherent anti-Stokes Raman scattering, second harmonic generation, and two photon excited fluorescence microscopy. Furthermore, a brain tumor specimen was additionally analyzed by linear Raman and Fourier transform infrared imaging for a detailed assessment of the tissue types that is required for classification and to validate the multimodal imaging approach. Hence label-free vibrational microspectroscopic imaging is a promising tool for fast and precise in vivo diagnostics of brain tumors.

Nonlinear Raman microspectroscopy based on a broadband coherent anti-Stokes Raman scattering is an emerging technique for noninvasive, chemically specific, microscopic analysis of tissues and large population of cells and particles. The sensitivity of this imaging is a critical aspect of a number of the proposed biomedical application. It is shown that the incident laser power is the major parameter controlling this sensitivity. By careful optimizing the laser system, the high-quality vibrational spectra acquisition at the multi-kHz rate becomes feasible.

The integration of living, human smooth muscle cells in biosynthesized cellulose scaffolds was monitored by nonlinear microscopy toward contractile artificial blood vessels. Combined coherent anti-Stokes Raman scattering (CARS) and second harmonic generation (SHG) microscopy was applied for studies of the cell interaction with the biopolymer network. CARS microscopy probing CH2-groups at 2845 cm^-1 permitted three-dimensional imaging of the cells with high contrast for lipid-rich intracellular structures. SHG microscopy visualized the fibers of the cellulose scaffold, together with a small signal obtained from the cytoplasmic myosin of the muscle cells. From the overlay images we conclude a close interaction between cells and cellulose fibers. We followed the cell migration into the three-dimensional structure, illustrating that while the cells submerge into the scaffold they extrude filopodia on top of the surface. A comparison between compact and porous scaffolds reveals a migration depth of <10 µm for the former, whereas the porous type shows cells further submerged into the cellulose. Thus, the scaffold architecture determines the degree of cell integration. We conclude that the unique ability of nonlinear microscopy to visualize the three-dimensional composition of living, soft matter makes it an ideal instrument within tissue engineering.

Coherent Raman microspectroscopy imaging is an emerging technique for noninvasive, chemically specific optical imaging, which can be potentially used to analyze the chemical composition and its distribution in biological tissues. In this report, a hierarchical cluster analysis was applied to hyperspectral coherent anti-Stokes Raman imaging of different chemical species through a turbid medium. It was demonstrated that by using readily available commercial software (Cytospec, Inc.) and cluster analysis, distinct chemical species can be imaged and identified through a rather thick layer of scattering medium. Once the clusters of different chemical composition were distinguished, a phase retrieval algorithm was used to convert coherent anti-Stokes Raman spectra to Raman spectra, which were used for chemical identification of hidden microscopic objects. In particular, applications to remote optical sensing of potential biological threats and to imaging through a layer of skin tissue were successfully demonstrated.

We demonstrate that doubly-resonant coherent anti-Stokes Raman scattering can provide enhanced and highly specific contrast for molecules containing unique Raman-active small molecular groups. This combination provides contrast for molecules that can otherwise be difficult to discriminate by Raman spectroscopy. Here, human monocytes were incubated with either deuterated oleic acid or 17-octadecynoic acid (a fatty acid with an end terminal acetylene group). The carbon-deuterium stretching vibration of the deuterated fatty acid, as well as the unique alkyne stretching vibration of the alkyne-containing fatty acid, were used to provide contrast for these exogenous free fatty acids. The combination of these unique modes with the common aliphatic carbon-hydrogen stretching vibration inherent to all fatty acid allowed for doubly-resonant detection of these unique molecules and enabled us to detect the presence of these lipids in areas within a cell where each molecular resonance by itself did not generate sufficient signal.

The development of methods that allow microscale studies of complex biomaterials based on their molecular composition is of great interest to a wide range of research fields. We show that stimulated Raman scattering (SRS) microscopy is an excellent analytical tool to study distributions of different biomolecules in multiphasic systems. SRS combines the label-free molecular specificity of vibrational spectroscopy with an enhanced sensitivity due to coherent excitation of molecular vibrations. Compared to previous imaging studies using coherent anti-Stokes Raman scattering microscopy, the main advantage of SRS microscopy is the absence of the unwanted nonresonant background, which translates into a superior sensitivity and undistorted vibrational spectra. We compare spectra of complex materials obtained with stimulated Raman scattering and spontaneous Raman scattering in the crowded fingerprint region. We find that, as expected, there is excellent correspondence and that the SRS spectra are free from interference from background fluorescence. In addition, we show high-resolution imaging of the distributions of selected biomolecules, such as lipids and proteins, in food products with SRS microscopy.

Experiments were conducted to study polarized light transmission in fresh bovine skeletal muscle of varying thicknesses. Two-dimensional polarization-sensitive transmission images were acquired and analyzed using a numerical parametric fitting algorithm. The total transmittance intensity and degree-of-polarization were calculated for both central ballistic and surrounding scattering regions. Full Mueller matrix images were derived from the raw polarization images and the polar decomposition algorithm was applied to extract polarization parameters. The results suggest that polarized light propagation through skeletal muscle is affected by strong birefringence, diattenuation, multiple scattering induced depolarization and the sarcomere diffraction effect.

This work describes a novel process for the fabrication of hybrid nanostructured particles showing intense tunable photoluminescence and a simultaneous ferromagnetic behavior. The fabrication process involves the synthesis of nanostructured porous silicon (NPSi) by chemical anodization of crystalline silicon and subsequent in pore growth of Co nanoparticles by electrochemically-assisted infiltration. Final particles are obtained by subsequent sonication of the Co-infiltrated NPSi layers and conjugation with poly(ethylene glycol) aiming at enhancing their hydrophilic character. These particles respond to magnetic fields, emit light in the visible when excited in the UV range, and internalize into human mesenchymal stem cells with no apoptosis induction. Furthermore, cytotoxicity in in-vitro systems confirms their biocompatibility and the viability of the cells after incorporation of the particles. The hybrid nanostructured particles might represent powerful research tools as cellular trackers or in cellular therapy since they allow combining two or more properties into a single particle.

Cancer is now recognized as one of the major causes of morbidity and mortality. Histopathological diagnosis, the gold standard, is shown to be subjective, time consuming, prone to interobserver disagreement, and often fails to predict prognosis. Optical spectroscopic methods are being contemplated as adjuncts or alternatives to conventional cancer diagnostics. The most important aspect of these approaches is their objectivity, and multivariate statistical tools play a major role in realizing it. However, rigorous evaluation of the robustness of spectral models is a prerequisite. The utility of Raman spectroscopy in the diagnosis of cancers has been well established. Until now, the specificity and applicability of spectral models have been evaluated for specific cancer types. In this study, we have evaluated the utility of spectroscopic models representing normal and malignant tissues of the breast, cervix, colon, larynx, and oral cavity in a broader perspective, using different multivariate tests. The limit test, which was used in our earlier study, gave high sensitivity but suffered from poor specificity. The performance of other methods such as factorial discriminant analysis and partial least square discriminant analysis are at par with more complex nonlinear methods such as decision trees, but they provide very little information about the classification model. This comparative study thus demonstrates not just the efficacy of Raman spectroscopic models but also the applicability and limitations of different multivariate tools for discrimination under complex conditions such as the multicancer scenario.

Instruments with single-molecule level detection capabilities can potentially benefit a wide variety of fields, including medical diagnostics. However, the size, cost, and complexity of such devices have prevented their widespread use outside sophisticated research laboratories. Fiber-only devices have recently been suggested as smaller and simpler alternatives, but thus far, they have lacked the resolution and sensitivity of a full-fledged system, and accurate alignment remains a critical requirement. Here we show that through-space reciprocal optical coupling between a fiber and a microscope objective, combined with wavelength division multiplexing in optical fibers, allows a drastic reduction of the size and complexity of such an instrument while retaining its resolution. We demonstrate a 4×4×18 cm³ sized fluorescence correlation spectrometer, which requires no alignment, can analyze kinetics at the single-molecule level, and has an optical resolution similar to that of much larger microscope based devices. The sensitivity can also be similar in principle, though in practice it is limited by the large background fluorescence of the commonly available optical fibers. We propose this as a portable and field deployable single molecule device with practical diagnostic applications.

We compare terahertz-pulsed imaging (TPI) with transverse microradiography (TMR) and microindentation to measure remineralization of artificial caries lesions. Lesions are formed in bovine enamel using a solution of 0.1 M lactic acid/0.2% Carbopol C907 and 50% saturated with hydroxyapatite adjusted to pH 5.0. The 20-day experimental protocol consists of four 1 min treatment periods with dentifrices containing 10, 675, 1385, and 2700 ppm fluoride, a 4-h/day acid challenge, and, for the remaining time, specimens are stored in a 50:50 pooled human/artificial saliva mixture. Each specimen is imaged at the focal point of the terahertz beam (data-point spacing = 50 μm). The time-domain data are used to calculate the refractive index volume percent profile throughout the lesion, and the differences in the integrated areas between the baseline and post-treatment profiles are used to calculate ΔΔZ(THz). In addition, the change from baseline in both the lesion depth and the intensity of the reflected pulse from the air/enamel interface is determined. Statistically significant Pearson correlation coefficients are observed between TPI and TMR/microindentation (P < 0.05). We demonstrate that TPI has potential as a research tool for hard tissue imaging.

In many fields of life science, visualization of spatial proximity, as an indicator of protein interactions in living cells, is of outstanding interest. A method to accomplish this is the measurement of Förster resonant energy transfer (FRET) by means of spectrally resolved fluorescence lifetime imaging microscopy. The fluorescence lifetime is calculated using a multiple-wavelength fitting routine. The donor profile is assumed first to have a monoexponential time-dependent behavior, and the acceptor decay profile is solved analytically. Later, the donor profile is assumed to have a two-exponential time-dependent behavior and the acceptor decay profile is derived analytically. We develop and apply a multispectral fluorescence lifetime imaging microscopy analysis system for FRET global analysis with time-resolved and spectrally resolved techniques, including information from donor and acceptor channels in contrast to using just a limited spectral data set from one detector only and a model accounting only for the donor signal. This analysis is used to demonstrate close vicinity of β-secretase (BACE) and GGA1, two proteins involved in Alzheimer's disease pathology. We attempt to verify if an improvement in calculating the donor lifetimes could be achieved when time-resolved and spectrally resolved techniques are simultaneously incorporated.

Emerging fields such as nanomedicine and nanotoxicology, demand new information on the effects of nanoparticles on biological membranes and lipid vesicles are suitable as an experimental model for bio-nano interaction studies. This paper describes image processing algorithms which stitch video sequences into mosaics and recording the shapes of thousands of lipid vesicles, which were used to assess the effect of CoFe₂O₄ nanoparticles on the population of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine lipid vesicles. The applicability of this methodology for assessing the potential of engineered nanoparticles to affect morphological properties of lipid membranes is discussed.

Microcirculation is an important component of the cardiovascular system and can be used to assess systemic cardiovascular health. Numerous studies have investigated cutaneous microcirculation as an indicator of cardiovascular related diseases. Such research has shown promising results; however, there are many limitations regarding the employed measurement techniques, such as poor depth and spatial resolution and measurement versatility. Here we show the results of functional cutaneous microvascular experiments measured with photoacoustic microscopy, which provides high spatial resolution and multiparameter measurements. In a set of experiments, microvascular networks located in the palms of volunteers were perturbed by periodic ischemic events, and the subsequent hemodynamic response to the stimulus was recorded. Results indicate that during periods of arterial occlusion, the relative oxygen saturation of the capillary vessels decreased below resting levels, and temporarily increased above resting levels immediately following the occlusion. Furthermore, a hyperemic reaction to the occlusions was measured, and the observation agreed well with similar measurements using more conventional imaging techniques. Due to its exceptional capability to functionally image vascular networks with high spatial resolution, photoacoustic microscopy could be a beneficial biomedical tool to assess microvascular functioning and applied to patients with diseases that affect cardiovascular health.

Laser speckle imaging (LSI) is a novel technique for measuring the mechanical properties of atherosclerotic plaques. In LSI, the decorrelation time constant of speckle intensity fluctuations provides an index of viscoelasticity that is closely related to plaque microstructure and composition. Here, we demonstrate for the first time, the feasibility of conducting LSI in vivo using a prototype 1.5 mm (4.5 Fr) diameter intravascular catheter. Investigation of the catheter performance using human arterial samples ex vivo shows that plaque time constants measured by the LSI catheter correlate well with those measured using a free-space bulk optics system. To demonstrate LSI in vivo, the catheter is interfaced with a portable console for intravascular evaluation in the aorta of a living rabbit. Distinct differences in arterial time constants are identified at normal aortic and stented sites in vivo with intravascular LSI.

We present the design and experimental results of a new MEMS-based endoscopic optical coherence tomography (OCT) probe. The uniqueness of this miniature OCT imaging probe is a two-axis MEMS mirror with through-silicon vias (TSVs) for interconnecting. The TSV interconnection enables ultracompact probe design, successfully reducing the probe size to only 2.6 mm in diameter. The MEMS mirror is actuated by an electrothermal actuator that is capable of scanning ±16° at only 3.6 V DC. Two-dimensional and three-dimensional OCT images of microspheres embedded in PDMS and acute rat brain tissue have been obtained with this miniature probe in a time-domain OCT system.

We present a novel, hand-held microscope probe for acquiring confocal images of biological tissue. This probe generates images by scanning a fiber-lens combination with a miniature electromagnetic actuator, which allows it to be operated in resonant and nonresonant scanning modes. In the resonant scanning mode, a circular field of view with a diameter of 190 μm and an angular frequency of 127 Hz can be achieved. In the nonresonant scanning mode, a maximum field of view with a width of 69 μm can be achieved. The measured transverse and axial resolutions are 0.60 and 7.4 μm, respectively. Images of biological tissue acquired in the resonant mode are presented, which demonstrate its potential for real-time tissue differentiation. With an outer diameter of 3 mm, the microscope probe could be utilized to visualize cellular microstructures in vivo across a broad range of minimally-invasive procedures.

Endogenous fluorescence provides morphological, spectral, and lifetime contrast that can indicate disease states in tissues. Previous studies have demonstrated that two-photon autofluorescence microscopy (2PAM) can be used for noninvasive, three-dimensional imaging of epithelial tissues down to approximately 150 μm beneath the skin surface. We report ex-vivo 2PAM images of epithelial tissue from a human tongue biopsy down to 370 μm below the surface. At greater than 320 μm deep, the fluorescence generated outside the focal volume degrades the image contrast to below one. We demonstrate that these imaging depths can be reached with 160 mW of laser power (2-nJ per pulse) from a conventional 80-MHz repetition rate ultrafast laser oscillator. To better understand the maximum imaging depths that we can achieve in epithelial tissues, we studied image contrast as a function of depth in tissue phantoms with a range of relevant optical properties. The phantom data agree well with the estimated contrast decays from time-resolved Monte Carlo simulations and show maximum imaging depths similar to that found in human biopsy results. This work demonstrates that the low staining inhomogeneity (∼20) and large scattering coefficient (∼10 mm−¹) associated with conventional 2PAM limit the maximum imaging depth to 3 to 5 mean free scattering lengths deep in epithelial tissue.

Foot ulceration remains a serious health concern for diabetic patients and has a major impact on the cost of diabetes treatment. Early detection and preventive care, such as offloading or improved hygiene, can greatly reduce the risk of further complications. We aim to assess the use of hyperspectral tissue oximetry in predicting the risk of diabetic foot ulcer formation. Tissue oximetry measurements are performed during several visits with hyperspectral imaging of the feet in type 1 and 2 diabetes mellitus subjects that are at risk for foot ulceration. The data are retrospectively analyzed at 21 sites that ulcerated during the course of our study and an ulceration prediction index is developed. Then, an image processing algorithm based on this index is implemented. This algorithm is able to predict tissue at risk of ulceration with a sensitivity and specificity of 95 and 80%, respectively, for images taken, on average, 58 days before tissue damage is apparent to the naked eye. Receiver operating characteristic analysis is also performed to give a range of sensitivity/specificity values resulting in a Q-value of 89%.

Direct noninvasive visualization of wound bed with depth information is important to understand the tissue repair. We correlate skin swept-source-optical coherence tomography (OCT) with histopathological and immunohistochemical evaluation on traumatic lower limb wounds under honey dressing to compare and assess the tissue repair features acquired noninvasively and invasively. Analysis of optical biopsy identifies an uppermost brighter band for stratum corneum with region specific thickness (p < 0.0001) and gray-level intensity (p < 0.0001) variation. Below the stratum corneum, variation in optical intensities is remarkable in different regions of the wound bed. Correlation between OCT and microscopic observations are explored especially in respect to progressive growth and maturation of the epithelial and subepithelial components. Characteristic transition of uniform hypolucid band in OCT image for depigmented zone to wavy highly lucid band in the pigmented zone could be directly correlated with the microscopic findings. The transformation of prematured epithelium of depigmented area, with low expression of E-cadherin, to matured epithelium with higher E-cadherin expression in pigmented zone, implicated plausible change in their optical properties as depicted in OCT. This correlated evaluation of multimodal images demonstrates applicability of swept-source-OCT in wound research and importance of integrated approach in validation of new technology.

Vulnerable plaques constitute a risk for serious heart problems, and are difficult to identify using existing methods. Hyperspectral imaging combines spectral- and spatial information, providing new possibilities for precise optical characterization of atherosclerotic lesions. Hyperspectral data were collected from excised aorta samples (n = 11) using both white-light and ultraviolet illumination. Single lesions (n = 42) were chosen for further investigation, and classified according to histological findings. The corresponding hyperspectral images were characterized using statistical image analysis tools (minimum noise fraction, K-means clustering, principal component analysis) and evaluation of reflectance/fluorescence spectra. Image analysis combined with histology revealed the complexity and heterogeneity of aortic plaques. Plaque features such as lipids and calcifications could be identified from the hyperspectral images. Most of the advanced lesions had a central region surrounded by an outer rim or shoulder-region of the plaque, which is considered a weak spot in vulnerable lesions. These features could be identified in both the white-light and fluorescence data. Hyperspectral imaging was shown to be a promising tool for detection and characterization of advanced atherosclerotic plaques in vitro. Hyperspectral imaging provides more diagnostic information about the heterogeneity of the lesions than conventional single point spectroscopic measurements.

Understanding the tumor microenvironment is critical to characterizing how cancers operate and predicting their response to treatment. We describe a novel, high-resolution coregistered photoacoustic (PA) and pulse echo (PE) ultrasound system used to image the tumor microenvironment. Compared to traditional optical systems, the platform provides complementary contrast and important depth information. Three mice are implanted with a dorsal skin flap window chamber and injected with PC-3 prostate tumor cells transfected with green fluorescent protein. The ensuing tumor invasion is mapped during three weeks or more using simultaneous PA and PE imaging at 25 MHz, combined with optical and fluorescent techniques. Pulse echo imaging provides details of tumor structure and the surrounding environment with 100-μm³ resolution. Tumor size increases dramatically with an average volumetric growth rate of 5.35 mm³/day, correlating well with 2-D fluorescent imaging (R = 0.97, p < 0.01). Photoacoustic imaging is able to track the underlying vascular network and identify hemorrhaging, while PA spectroscopy helps classify blood vessels according to their optical absorption spectrum, suggesting variation in blood oxygen saturation. Photoacoustic and PE imaging are safe, translational modalities that provide enhanced depth resolution and complementary contrast to track the tumor microenvironment, evaluate new cancer therapies, and develop molecular contrast agents in vivo.

G-protein-coupled receptors are important targets for various drugs. After signal transduction, regulatory processes, such as receptor desensitization and internalization, change the lateral receptor mobility. In order to study the lateral diffusion of β_2-adrenergic receptors (β₂AR) complexed with fluorescently labeled noradrenaline (Alexa-NA) in plasma membranes of A549 cells, trajectories of single receptor-ligand complexes were monitored using single-particle tracking. We found that a fraction of 18% of all β₂ARs are constitutively immobile. About 2/3 of the β₂ARs moved with a diffusion constant of D₂ = 0.03±0.001 μm²/s and about 17% were diffusing five-fold faster (D₃ = 0.15±0.02 μm²/s). The mobile receptors moved within restricted domains and also showed a discontinuous diffusion behavior. Analysis of the trajectory lengths revealed two different binding durations with τ₁ = 77±1 ms and τ₂ = 388±11 ms. Agonistic stimulation of the β₂AR-Alexa-NA complexes with 1 μM terbutaline caused immobilization of almost 50% of the receptors within 35 min. Simultaneously, the mean area covered by the mobile receptors decreased significantly. Thus, we demonstrated that agonistic stimulation followed by cell regulatory processes results in a change in β₂AR mobility suggesting that different receptor dynamics characterize different receptor states.

Many interferometry-based quantitative phase contrast imaging techniques require a separately generated coherent reference wave. This results in a low phase stability and the demand for a precise adjustment of the intensity ratio between object and reference wave. To overcome these problems, the performance of a Michelson interferometer approach for digital holographic microscopy was analyzed that avoids a separately generated reference wave by superposition of different image areas. It is shown that this simplified arrangement yields improved phase stability. Furthermore, results from time-lapse investigations on living pancreas tumor cells demonstrate the capability of the method for reliable quantitative phase contrast imaging.

The influence of different tissue preservation (a test solution under development and a standard storage solution) on human cornea morphology, refractive index and hydration was assessed noninvasively by ultrahigh-resolution optical coherence tomography (OCT) over time. For 28 days' or 15 days' storage in the preservation media, corneas in the two media exhibited different structural changes with different onset times including epithelial desquamation, edema-induced cornea thickening and change in tissue refractive index. It was found that the variation of the group refractive index over time was only about 2%, while 25% variation of hydration was observed in the storage and subsequent return to normothermic conditions in both preservation media. The results suggest the two media involved different but correlated preservation mechanisms. This study demonstrates that the noncontact, noninvasive, and high-resolution OCT is a powerful tool for noninvasive characterization of tissue morphological changes and hydration process and for assessment of the effects of preservation media on stored tissue integrity.

Previous studies have shown the ability of many lymphatic vessels to contract phasically to pump lymph. Every lymphangion can act like a heart with pacemaker sites that initiate the phasic contractions. The contractile wave propagates along the vessel to synchronize the contraction. However, determining the location of the pacemaker sites within these vessels has proven to be very difficult. A high speed video microscopy system with an automated algorithm to detect pacemaker location and calculate the propagation velocity, speed, duration, and frequency of the contractions is presented in this paper. Previous methods for determining the contractile wave propagation velocity manually were time consuming and subject to errors and potential bias. The presented algorithm is semiautomated giving objective results based on predefined criteria with the option of user intervention. The system was first tested on simulation images and then on images acquired from isolated microlymphatic mesenteric vessels. We recorded contraction propagation velocities around 10 mm/s with a shortening speed of 20.4 to 27.1 μm/s on average and a contraction frequency of 7.4 to 21.6 contractions/min. The simulation results showed that the algorithm has no systematic error when compared to manual tracking. The system was used to determine the pacemaker location with a precision of 28 μm when using a frame rate of 300 frames per second.

Currently no standard clinical or preclinical noninvasive method exists to monitor cell death based on morphological changes at the cellular level. In our past work we have demonstrated that quantitative high frequency ultrasound imaging can detect cell death in vitro and in vivo. In this study we apply quantitative methods previously used with high frequency ultrasound to optical coherence tomography (OCT) to detect cell death. The ultimate goal of this work is to use these methods for optically-based clinical and preclinical cancer treatment monitoring. Optical coherence tomography data were acquired from acute myeloid leukemia cells undergoing three modes of cell death. Significant increases in integrated backscatter were observed for cells undergoing apoptosis and mitotic arrest, while necrotic cells induced a decrease. These changes appear to be linked to structural changes observed in histology obtained from the cell samples. Signal envelope statistics were analyzed from fittings of the generalized gamma distribution to histograms of envelope intensities. The parameters from this distribution demonstrated sensitivities to morphological changes in the cell samples. These results indicate that OCT integrated backscatter and first order envelope statistics can be used to detect and potentially differentiate between modes of cell death invitro.

High-speed fluorescence molecular tomography (FMT) reconstruction for 3-D heterogeneous media is still one of the most challenging problems in diffusive optical fluorescence imaging. In this paper, we propose a fast FMT reconstruction method that is based on Monte Carlo (MC) simulation and accelerated by a cluster of graphics processing units (GPUs). Based on the Message Passing Interface standard, we modified the MC code for fast FMT reconstruction, and different Green's functions representing the flux distribution in media are calculated simultaneously by different GPUs in the cluster. A load-balancing method was also developed to increase the computational efficiency. By applying the Fréchet derivative, a Jacobian matrix is formed to reconstruct the distribution of the fluorochromes using the calculated Green's functions. Phantom experiments have shown that only 10 min are required to get reconstruction results with a cluster of 6 GPUs, rather than 6 h with a cluster of multiple dual opteron CPU nodes. Because of the advantages of high accuracy and suitability for 3-D heterogeneity media with refractive-index-unmatched boundaries from the MC simulation, the GPU cluster-accelerated method provides a reliable approach to high-speed reconstruction for FMT imaging.

We show that applying the Laplace operator to a speckle-free quantitative phase image reveals an unprecedented level of detail in cell structure, without the gradient artifacts associated with differential interference contrast microscopy, or photobleaching and phototoxicity limitations common in fluorescence microscopy. This method, referred to as Laplace phase microscopy, is an efficient tool for tracking vesicles and organelles in living cells. The principle is demonstrated by tracking organelles in cardiomyocytes and vesicles in neurites of hippocampal neurons, which to our knowledge are the first label-free diffusion measurements of the organelles in such cells.

Saccharide interferences such as Dextran, Galactose, etc. have a great potential to interfere with near infrared (NIR) glucose analysis since they have a similar spectroscopic fingerprint and are present physiologically at large relative concentrations. These can lead to grossly inappropriate interpretation of patient glucose levels and resultant treatment in critical care and hospital settings. This study describes a methodology to reduce this effect on glucose analysis using an NIR Fourier transform spectroscopy method combined with a multivariate calibration technique (PLS) using preprocessing by orthogonal signal correction (OSC). A mathematical approach based on the use of a single calibration based bias and slope correction was applied in addition to a standard OSC was investigated. This approach is combined with a factorial interferent calibration design to accommodate for interference effects. We named this approach as a slope and bias OSC (sbOSC). sbOSC differs from OSC in the way it handles the prediction. In sbOSC, statistics on slope and bias obtained from a set of calibration samples are then used as a validation parameter in the prediction set. Healthy human volunteer blood with different glucose (80 to 200 mg/dL) and hematocrit (24 to 48 vol.%) levels containing high expected levels of inteferents have been measured with a transmittance near-infrared Fourier transform spectrometer operates in the broadband spectral range of 1.25-2.5 μm (4000-8000 cm−¹). The effect of six interferents compounds used in intensive care and operating rooms, namely Dextran, Fructose, Galactose, Maltose, Mannitol, and Xylose, were tested on blood glucose. A maximum interference effect (MIE) parameter was used to rank the significance for the individual interferent type on measurement error relative to the total NIR whole blood glucose measurement error. For comparison, a YSI (Yellow Springs Instrument) laboratory reference glucose analyzer and NIR data were collected at the same time as paired samples.

A light-scattering signal, which is sensitive to cellular/subcellular structural integrity, is a potential indicator of brain tissue viability because metabolic energy is used in part to maintain the structure of cells. We previously observed a unique triphasic scattering change (TSC) at a certain time after oxygen/glucose deprivation for blood-free rat brains; TSC almost coincided with the cerebral adenosine triphosphate (ATP) depletion. We examine whether such TSC can be observed in the presence of blood in vivo, for which transcranial diffuse reflectance measurement is performed for rat brains during hypoxia induced by nitrogen gas inhalation. At a certain time after hypoxia, diffuse reflectance intensity in the near-infrared region changes in three phases, which is shown by spectroscopic analysis to be due to scattering change in the tissue. During hypoxia, rats are reoxygenated at various time points. When the oxygen supply is started before TSC, all rats survive, whereas no rats survive when the oxygen supply is started after TSC. Survival is probabilistic when the oxygen supply is started during TSC, indicating that the period of TSC can be regarded as a critical time zone for rescuing the brain. The results demonstrate that light scattering signal can be an indicator of brain tissue reversibility.

Raman spectroscopy was used to study temporal molecular changes associated with spinal cord injury (SCI) in a rat model. Raman spectra of saline-perfused, injured, and healthy rat spinal cords were obtained and compared. Two injury models, a lateral hemisection and a moderate contusion were investigated. The net fluorescence and the Raman spectra showed clear differences between the injured and healthy spinal cords. Based on extensive histological and biochemical characterization of SCI available in the literature, these differences were hypothesized to be due to cell death, demyelination, and changes in the extracellular matrix composition, such as increased expression of proteoglycans and hyaluronic acid, at the site of injury where the glial scar forms. Further, analysis of difference spectra indicated the presence of carbonyl containing compounds, hypothesized to be products of lipid peroxidation and acid catalyzed hydrolysis of glycosaminoglycan moieties. These results compared well with in vitro experiments conducted on chondroitin sulfate sugars. Since the glial scar is thought to be a potent biochemical barrier to nerve regeneration, this observation suggests the possibility of using near infrared Raman spectroscopy to study injury progression and explore potential treatments exvivo, and ultimately monitor potential remedial treatments within the spinal cord in vivo.

Arterial revascularization in patients with peripheral arterial disease (PAD) reestablishes large arterial blood supply to the ischemic muscles in lower extremities via bypass grafts or percutaneous transluminal angioplasty (PTA). Currently no gold standard is available for assessment of revascularization effects in lower extremity muscles. This study tests a novel near-infrared diffuse correlation spectroscopy flow-oximeter for monitoring of blood flow and oxygenation changes in medial gastrocnemius (calf) muscles during arterial revascularization. Twelve limbs with PAD undergoing revascularization were measured using a sterilized fiber-optic probe taped on top of the calf muscle. The optical measurement demonstrated sensitivity to dynamic physiological events, such as arterial clamping/releasing during bypass graft and balloon inflation/deflation during PTA. Significant elevations in calf muscle blood flow were observed after revascularization in patients with bypass graft (+48.1 ± 17.5%) and patients with PTA (+43.2 ± 11.0%), whereas acute post-revascularization effects in muscle oxygenation were not evident. The decoupling of flow and oxygenation after revascularization emphasizes the need for simultaneous measurement of both parameters. The acute elevations/improvements in calf muscle blood flow were associated with significant improvements in symptoms and functions. In total, the investigation corroborates potential of the optical methods for objectively assessing the success of arterial revascularization.

Orthotopic bladder cancer model in rats mimics human bladder cancer with respect to urothelial tumorigenesis and progression. Utilizing this model at pT1 (superficial stage), we analyze the tissue responses to hexyl 5-aminolevulinate-induced photodynamic therapy (HAL-PDT). In comparison to untreated rats, HAL-PDT causes little change in tumor-free rat bladder but induces inflammatory changes with increased lymphocytes and mononuclear cell infiltration in rat bladders with tumor. Immunohistochemistry reveals that HAL-PDT is without effect on proliferating cell nuclear antigen expression within the tumor and increases caspase-3 expression in both normal urothelium and the tumor. Transmission electron microscopy reveals severe mitochondrial damage, formations of apoptotic bodies, vacuoles, and lipofuscin bodies, but no microvillus-formed niches in HAL-PDT-treated bladder cancer rats. Bioinformatics analysis of the gene expression profile indicates an activation of T-cell receptor signaling pathway in bladder cancer rats without PDT. HAL-PDT increases the expression of CD3 and CD45RA in the tumor (determined by immunohistochemistry). We suggest that pathways of action of HAL-PDT may include, at least, activations of mitochondrial apoptosis and autophagy, breakdown of cancer stem cell niches, and importantly, enhancement of T-cell activation.

Optical transfection is a powerful method for targeted delivery of therapeutic agents to biological cells. A tightly focused pulsed laser beam may transiently change the permeability of a cell membrane to facilitate the delivery of foreign genetic material into cells. We report the first realization of an endoscope-like integrated system for optical transfection. An imaging fiber (coherent optical fiber bundle) with ∼6000 cores (pixels) embedded in a fiber cladding of ∼300 μm in diameter, produces an image circle (area) of ∼270 μm diam. This imaging fiber, with an ordered axicon lens array chemically etched at its exit face, is used for the delivery of a femtosecond laser to the cell membrane for optical transfection along with subcellular resolution imaging. A microcapillary-based microfluidic system for localized drug delivery was also combined in this miniature, flexible system. Using this novel system, a plasmid transfection efficiency up to ∼72% was obtained for CHO-K1 cells. This endoscope-like system opens a range of exciting applications, in particular, in the targeted in vivo optical microsurgery area.

Previous works investigated a spectroscopic technique that offered a promising alternative to blood and breath assays for determining invivo alcohol concentration. Although these prior works measured the dorsal forearm, we report the results of a 26-subject clinical study designed to evaluate the spectroscopic technique at a finger measurement site through comparison to contemporaneous forearm spectroscopic, venous blood, and breath measurements. Through both Monte Carlo simulation and experimental data, it is shown that tissue optical probe design has a substantial impact on the effective path-length of photons through the skin and the signal-to-noise ratio of the spectroscopic measurements. Comparison of the breath, blood, and tissue assays demonstrated significant differences in alcohol concentration that are attributable to both assay accuracy and alcohol pharmacokinetics. Similar to past works, a first order kinetic model is used to estimate the fraction of concentration variance explained by alcohol pharmacokinetics (72.6-86.7%). A significant outcome of this work was significantly improved pharmacokinetic agreement with breath (arterial) alcohol of the finger measurement (mean kArt-Fin = 0.111 ^min−1) relative to the forearm measurement (mean kArt-For = 0.019 ^min−1) that is likely due to the increased blood perfusion of the finger.

Decreasing the pulse duration helps confine damage, shorten treatment time, and minimize pain during retinal photocoagulation. However, the safe therapeutic window (TW), the ratio of threshold powers for thermomechanical rupture of Bruch's membrane and mild coagulation, also decreases with shorter exposures. Two potential approaches toward increasing TW are investigated: (a) decreasing the central irradiance of the laser beam and (b) temporally modulating the pulse. An annular beam with adjustable central irradiance was created by coupling a 532-nm laser into a 200-μm core multimode optical fiber at a 4-7 deg angle to normal incidence. Pulse shapes were optimized using a computational model, and a waveform generator was used to drive a PASCAL photocoagulator (532 nm), producing modulated laser pulses. Acute thresholds for mild coagulation and rupture were measured in Dutch-Belted rabbit in vivo with an annular beam (154-163 μm retinal diameter) and modulated pulse (132 μm, uniform irradiance "flat-top" beam) with 2-50 ms pulse durations. Thresholds with conventional constant-power pulse and a flat-top beam were also determined. Both annular beam and modulated pulse provided a 28% increase in TW at 10-ms duration, affording the same TW as 20-ms pulses with conventional parameters.