Vertebrate retinal rod outer segment disks house the proteins involved in the phototransduction cascade that converts light into neuronal signal. We develop a technique for the immunofluorescent labeling of osmotically intact isolated rod outer segment disks for confocal laser scanning microscopy imaging. Osmotically intact Ficoll-flotation isolated bovine disks are directly labeled with antibodies in solution. For the first time, osmotically intact single disks can be visualized. Thus, imaging of purified disks, based on advanced optical techniques, may serve as a powerful complement to other methods in studies on phototransduction. In fact, even though much is known about the rod outer segment photoresponse, some unanswered questions remain, particularly about ATP supply, light adaptation, and morphogenesis.

Two-photon microscopy has been used to perform high spatial resolution imaging of spine plasticity in the intact neocortex of living mice. Multiphoton absorption has also been used as a tool for the selective disruption of cellular structures in living cells and simple organisms. In this work, we exploit the spatial localization of multiphoton excitation to perform selective lesions on the neuronal processes of cortical neurons in living mice expressing fluorescent proteins. Neurons are irradiated with a focused, controlled dose of femtosecond laser energy delivered through cranial optical windows. The morphological consequences are then characterized with time lapse 3-D two-photon imaging over a period of minutes to days after the procedure. This methodology is applied to dissect single dendrites with submicrometric precision without causing any visible collateral damage to the surrounding neuronal structures. The spatial precision of this method is demonstrated by ablating individual dendritic spines, while sparing the adjacent spines and the structural integrity of the dendrite. The combination of multiphoton nanosurgery and in vivo imaging in mammals represents a promising tool for neurobiology and neuropharmacology research.

We present the first experimental result of direct delineation of the nuclei of living rat bladder epithelium with ultrahigh-resolution optical coherence tomography (uOCT). We demonstrate that the cellular details embedded in the speckle noise in a uOCT image can be uncovered by time-lapse frame averaging that takes advantage of the micromotion in living biological tissue. The uOCT measurement of the nuclear size (7.9±1.4 μm) closely matches the histological evaluation (7.2±0.8 μm). Unlike optical coherence microscopy (OCM), which requires a sophisticated high-NA microscopic objective, this approach uses a commercial-grade single achromatic lens (f/10 mm, NA/0.25) and provides a cross-sectional image over 0.6 mm of depth without focus tracking, thus holding great promise of endoscopic optical biopsy for diagnosis and grading of flat epithelial cancer such as carcinoma in situ in vivo.

This PDF file contains the editorial “Guest Editorial: Optical Diagnostic Imaging from Bench to Bedside” for JBO Vol. 12 Issue 05

Barrett's esophagus (BE) is a condition that poses high risk of developing dysplasia leading to cancer. Detection of dysplasia is a critical element in determining therapy but is extremely challenging, so that standard white-light endoscopy is used only as a means to guide biopsy. Many novel optical techniques have been aimed at this problem, including various forms of improved wide-field white-light (chromoendscopy/magnification and narrow-band) and fluorescence imaging, and "optical biopsy" techniques (diffuse reflectance, elastic light scattering, fluorescence and Raman spectroscopies, confocal microendoscopy, and optical coherence tomography). While promising, either as stand-alone modalities or in combination, to date none has solved this pivotal challenge to the point of clinical adoption. Likewise, minimally invasive treatment of BE patients with dysplasia remains suboptimal, despite recent approval of photodynamic therapy for this indication. This work presents a critique and summary of each of these biophotonic technologies, and discusses the fundamental advantages and limitations of each. The future directions for this field are considered, particularly from the perspective of relying on intrinsic (endogenous) optical signatures compared with the use of exogenous contrast agents.

Optical brain imaging has seen 30 years of intense development, and has grown into a rich and diverse field. In-vivo imaging using light provides unprecedented sensitivity to functional changes through intrinsic contrast, and is rapidly exploiting the growing availability of exogenous optical contrast agents. Light can be used to image microscopic structure and function in vivo in exposed animal brain, while also allowing noninvasive imaging of hemodynamics and metabolism in a clinical setting. This work presents an overview of the wide range of approaches currently being applied to in-vivo optical brain imaging, from animal to man. Techniques include multispectral optical imaging, voltage sensitive dye imaging and speckle-flow imaging of exposed cortex, in-vivo two-photon microscopy of the living brain, and the broad range of noninvasive topography and tomography approaches to near-infrared imaging of the human brain. The basic principles of each technique are described, followed by examples of current applications to cutting-edge neuroscience research. In summary, it is shown that optical brain imaging continues to grow and evolve, embracing new technologies and advancing to address ever more complex and important neuroscience questions.

Since its introduction, optical coherence tomography (OCT) technology has advanced from the laboratory bench to the clinic and back again. Arising from the fields of low coherence interferometry and optical time- and frequency-domain reflectometry, OCT was initially demonstrated for retinal imaging and followed a unique path to commercialization for clinical use. Concurrently, significant technological advances were brought about from within the research community, including improved laser sources, beam delivery instruments, and detection schemes. While many of these technologies improved retinal imaging, they also allowed for the application of OCT to many new clinical areas. As a result, OCT has been clinically demonstrated in a diverse set of medical and surgical specialties, including gastroenterology, dermatology, cardiology, and oncology, among others. The lessons learned in the clinic are currently spurring a new set of advances in the laboratory that will again expand the clinical use of OCT by adding molecular sensitivity, improving image quality, and increasing acquisition speeds. This continuous cycle of laboratory development and clinical application has allowed the OCT technology to grow at a rapid rate and represents a unique model for the translation of biomedical optics to the patient bedside. This work presents a brief history of OCT development, reviews current clinical applications, discusses some clinical translation challenges, and reviews laboratory developments poised for future clinical application.

The wavelength resolved spectral fluorescence imaging technique using a fluorescein-conjugated avidin has been reported to visualize submillimeter implants of ovarian cancer because of its highly targeted and quickly cleared pharmacokinetics. However, clinical application of avidin was hampered by its strong immunogenicity. As a clinically feasible alternative to avidin, which targets the same D-galactose receptor but is made from a nonimmunogenic source, with even better binding capability by multiplying binding sites but still maintaining a favorable characteristic of high isoelectric point, a serum albumin conjugated with 23 galactosamine and 2 rhodamine green molecules (GmSA-RhodG) was designed and synthesized. GmSA-RhodG showed more than 10-fold rapid and higher uptake by SHIN3 ovarian cancer cells than both avidin- and no galactosamine-conjugated albumin (bovine serum)–RhodG. Sensitivity and specificity of GmSA-RhodG to detect red fluorescence labeled peritoneal cancer foci in mouse cancer model were 100%/99% (n=566), respectively for 1-~mm lesions and even smaller lesions were detected in vivo. These results indicate that GmSA-RhodG is not only a clinically feasible alternative but more efficient targeting reagent for D-galactose receptors than avidin-RhodG.

The Imaging Probe Development Center (IPDC) has been set up under the auspices of the National Institutes of Health (NIH) Roadmap as part of the molecular libraries and imaging initiatives. It comprises a core synthesis facility dedicated to the preparation of imaging probes, initially for intramural NIH scientists, and later, for the extramural scientific community. The facility opened fully in late 2006, in refurbished laboratories in Rockville, Maryland, and a staff of around a dozen was recruited into place by early 2007; the director was hired in late 2005. The IPDC provides a mechanism for the production of sensitive probes for use by imaging scientists who cannot obtain such probes commercially. The probes to be made will encompass all major imaging modalities including radionuclide, magnetic resonance, and optical. The operation of the IPDC is outlined, together with the results of interim achievements while the IPDC maintained a small temporary laboratory in Bethesda. As of December 2006, a total of eleven probe compositions had been made, and several of these are described with particular mention of those probes intended for use in optical applications.

The goal of this work is to develop in vivo photoacoustic (PA) flow cytometry (PAFC) for time-resolved detection of circulating absorbing objects, either without labeling or with nanoparticles as PA labels. This study represents the first attempt, to our knowledge, to demonstrate the capability of PAFC with tunable near-infrared (NIR) pulse lasers for real-time monitoring of gold nanorods, Staphylococcus aureus and Escherichia coli labeled with carbon nanotubes (CNTs), and contrast dye Lymphazurin in the microvessels of mouse and rat ears and mesenteries. PAFC shows the unprecedented threshold sensitivity in vivo as one gold nanoparticle in the irradiated volume and as one bacterium in the background of 10⁸ of normal blood cells. The CNTs are demonstrated to serve as excellent new NIR high-PA contrast agents. Fast Lymphazurin diffusion in live tissue is observed with rapid blue coloring of a whole animal body. The enhancement of the thermal and acoustic effects is obtained with clustered, multilayer, and exploded nanoparticles.This novel combination of PA microscopy/spectroscopy and flow cytometry may be considered as a new powerful tool in biological research with the potential of quick translation to humans, providing ultrasensitive diagnostics of pathogens (e.g., bacteria, viruses, fungi, protozoa, parasites, helminthes), metastatic, infected, inflamed, stem, and dendritic cells, and pharmacokinetics of drug, liposomes, and nanoparticles in deep vessels (with focused transducers) among other potential applications.

Iron oxide particles are becoming an important contrast agent for magnetic resonance imaging (MRI) cell tracking studies. Simultaneous delivery of fluorescence indicators with the particles to individual cells offers the possibility of correlating optical images and MRI. In this work, it is demonstrated that micron-sized iron oxide particles (MPIOs) can be used as a carrier to deliver fluorescent probes to cells in culture as well as to migrating neural progenitors in vivo. Migrating progenitors were tracked with MRI and easily identified by histology because of the fluorescent probe. These data suggest that using MPIOs to deliver fluorescent probes should make it possible to combine MRI and optical imaging for in vivo cell tracking.

Metallic nanoparticles have unique optical properties that can be exploited for molecular imaging in tissue. Image contrast depends on the nature of the particles, properties of the target tissue, and the imaging system. Maximizing image contrast for a particular application requires an understanding of the interplay of these factors. We demonstrate an approach that integrates the use of reflectance spectroscopy and imaging of particles in water and various tissue phantoms to evaluate the expected image contrast. We illustrate the application of this methodology for gold and silver nanospheres targeted against a biomarker expressed in epithelial tissue; predictions of contrast properties using diffuse reflectance spectroscopy were compared with widefield and high-resolution images of labeled tissue phantoms. The results show that the predicted image contrast based on spectroscopy agrees well with widefield and high-resolution imaging, and illustrate that gold and silver nanospheres at subnanomolar concentration are sufficient to produce contrast in both imaging modes. However, the effective contrast achieved with a particular type of nanoparticle can differ dramatically depending on the imaging modality. The ability to predict and optimize image contrast properties is a crucial step in the effective use of these nanomaterials for biomedical imaging applications.

In a previous study, we investigated physical methods to reduce whole-body, diet-related autofluorescence interference in several mouse strains through changes in animal diet. Measurements of mice with an in vivo multispectral imaging system over a 21-day period allowed for the quantification of concentration changes in multiple in vivo fluorophores. To be an effective instrument, a multispectral imaging system requires a priori spectral knowledge, the form and importance of which is not necessarily intuitive, particularly when noninvasive in vivo longitudinal imaging studies are performed. Using an optimized spectral library from a previous autofluorescence-reduction study as a model, we investigated two additional spectral definition techniques to illustrate the results of poor spectral definition in a longitudinal fluorescence imaging study. Here we systematically evaluate these results and show how poor spectral definition can lead to physiologically irrelevant results. This study concludes that the proper selection of robust spectra corresponding to each specific fluorescent molecular label of interest is of integral importance to enable effective use of multispectral imaging techniques in longitudinal fluorescence studies.

We present an experimental test of a new spectral approach that is aimed at quantifying the relative concentrations of two chromophores that are contained in a defect embedded in a turbid medium. The basic steps of our spectral approach are (a) perform a linear tandem scan of the source and detector across the defect; (b) measure the spectral dependence of the maximum change induced by the defect in the scanned intensity; (c) identify a set of appropriate pairs of wavelengths (λ1, λ2) at which such maximum intensity changes are the same; and (d) measure the reduced scattering coefficient spectrum of the background medium. For each wavelength pair (λ1, λ2), we obtain a measurement of the relative concentrations of the two chromophores, where the only required parameters are the extinction coefficients of the two chromophores and the ratio of the background scattering coefficients at λ1 and λ2. In a mixture of two test chromophores (blue food coloring dye and black India ink) contained in a 0.78-cm diameter cylinder, our spectral approach yielded relative concentrations values that were within 6% of their actual values. Although our paired-wavelength spectral approach is not generally applicable to any pair of chromophores, it is suitable for oxyhemoglobin and deoxyhemoglobin and is thus appropriate for oximetry of localized lesions in biological tissues.

Typical manifestations of cutaneous inflammation include erythema and edema. While erythema is the result of capillary dilation and local increase of oxygenated hemoglobin concentration, edema is characterized by an increase in extracellular fluid in the dermis, leading to local tissue swelling. Both of these inflammatory reactions are typically graded visually. We demonstrate the potential of spectral imaging as an objective noninvasive method for quantitative documentation of both erythema and edema. As examples of dermatological conditions that exhibit skin inflammation we applied this method on patients suffering from (1) allergic dermatitis (poison ivy rashes), (2) inflammatory acne, and (3) viral infection (herpes zoster). Spectral images are acquired in the visible and near-IR part of the spectrum. Based on a spectral decomposition algorithm, apparent concentrations maps are constructed for oxyhemoglobin, deoxyhemoglobin, melanin, optical scattering, and water. In each dermatological condition examined, the concentration maps of oxyhemoglobin and water represent quantitative visualizations of the intensity and extent of erythema and cutaneous edema, correspondingly. We demonstrate that spectral imaging can be used to quantitatively document parameters relevant to skin inflammation. Applications may include monitoring of disease progression as well as screening for efficacy of treatments.

This research describes a noninvasive, noncontact method used to quantitatively analyze the functional characteristics of tissue. Multispectral images collected at several near-infrared wavelengths are input into a mathematical optical skin model that considers the contributions from different analytes in the epidermis and dermis skin layers. Through a reconstruction algorithm, we can quantify the percent of blood in a given area of tissue and the fraction of that blood that is oxygenated. Imaging normal tissue confirms previously reported values for the percent of blood in tissue and the percent of blood that is oxygenated in tissue and surrounding vasculature, for the normal state and when ischemia is induced. This methodology has been applied to assess vascular Kaposi's sarcoma lesions and the surrounding tissue before and during experimental therapies. The multispectral imaging technique has been combined with laser Doppler imaging to gain additional information. Results indicate that these techniques are able to provide quantitative and functional information about tissue changes during experimental drug therapy and investigate progression of disease before changes are visibly apparent, suggesting a potential for them to be used as complementary imaging techniques to clinical assessment.

Optical coherence tomography (OCT) is a micron scale high-resolution optical technology that can provide real-time in vivo images noninvasively. The ability to detect airway mucosal and submucosal injury rapidly will be valuable for a range of pulmonary applications including assessment of acute inhalation smoke and burn injury. OCT has the potential ability to monitor the progression of airway injury changes including edema, hyperemia, and swelling, which are critical clinical components of smoke-inhalation injury. New Zealand white male rabbits exposed to cold smoke from standardized unbleached burned cotton administered during ventilation were monitored for 6 h using a 1.8-mm diameter flexible fiberoptic longitudinal probe that was inserted through the endotracheal tube. The thickness of the epithelial, mucosal, and submucosal layers of the rabbit trachea to the tracheal cartilage was measured using a prototype superluminescent diode OCT system we constructed. OCT was able to detect significant smoke-injury-induced increases in the thickness of the tracheal walls of the rabbit beginning very shortly after smoke administration. Airway wall thickness increased to an average of 120% (±33%) of baseline values by 5 h following exposure. OCT is capable of providing real-time, noninvasive images of airway injury changes following smoke exposure. These studies suggest that OCT may have the ability to provide information on potential early indicators of impending smoke-inhalation-induced airway compromise.

A preliminary study to assess noninvasive optical coherence tomography (OCT) for early detection and evaluation of chemotherapy-induced oral mucositis in five patients. In five patients receiving neoadjuvant chemotherapy for primary breast cancer, oral mucositis was assessed clinically, and imaged using noninvasive OCT. Imaging was scored using a novel imaging-based scoring system. Conventional clinical assessment using the Oral Mucositis Assessment Scale was used as the gold standard. Patients were evaluated on days 0, 2, 4, 7, and 11 after commencement of chemotherapy. OCT images were visually examined by one blinded investigator. The following events were identified using OCT: (1) change in epithelial thickness and subepithelial tissue integrity (beginning on day 2), (2) loss of surface keratinized layer continuity (beginning on day 4), (3) loss of epithelial integrity (beginning on day 4). Imaging data gave higher scores compared to clinical scores earlier in treatment, suggesting that the imaging-based diagnostic scoring was more sensitive to early mucositic change than the clinical scoring system. Once mucositis was established, imaging and clinical scores converged. Chemotherapy-induced oral changes were identified prior to their clinical manifestation using OCT, and the proposed scoring system for oral mucositis was validated for the semiquantification of mucositic change.

A series of bench to operating room studies was conducted to determine whether it is feasible to use optical coherence tomography (OCT) clinically to diagnose potentially reversible early cartilage degeneration. A human cadaver study was performed to confirm the reproducibility of OCT imaging and grading based on identification of changes to cartilage OCT form birefringence using a polarized OCT system approved for clinical use. Segregation of grossly normal appearing human articular cartilage into two groups based on the presence or absence of OCT form birefringence showed that cartilage without OCT form birefringence had reduced ability to increase proteoglycan synthetic activity in response to the anabolic growth factor IGF-1. The bench data further show that IGF-1 insensitivity in cartilage without OCT form birefringence was reversible. To show clinical feasibility, OCT was then used arthroscopically in 19 human subjects. Clinical results confirmed that differences to OCT form birefringence observed in ex vivo study were detectable during arthroscopic surgery. More prevalent loss of cartilage OCT form birefringence was observed in cartilage of human subjects in groups more likely to have cartilage degeneration. This series of integrated bench to bedside studies demonstrates translational feasibility to use OCT for clinical studies on whether human cartilage degeneration can be diagnosed early enough for intervention that may delay or prevent the onset of osteoarthritis.

Recent advances in catheter-based optical coherence tomography (OCT) have provided the necessary resolution and acquisition speed for high-quality intravascular imaging. Complications associated with clearing blood from the vessel of a living patient have prevented its wider acceptance. We identify a surgical application that takes advantage of the vascular imaging powers of OCT but that circumvents the difficulties. Coronary artery bypass grafting (CABG) is the most commonly performed major surgery in America. A critical determinant of its outcome has been postulated to be injury to the conduit vessel incurred during the harvesting procedure or pathology preexistent in the harvested vessel. As a test of feasibility, intravascular OCT imaging is obtained from the radial arteries (RAs) and/or saphenous veins (SVs) of 35 patients scheduled for CABG. Pathologies detected by OCT are compared to registered histological sections obtained from discarded segments of each graft. OCT reliably detects atherosclerotic lesions in the RAs and discerns plaque morphology as fibrous, fibrocalcific, or fibroatheromatous. OCT is also used to assess intimal trauma and residual thrombi related to endoscopic harvest and the quality of the distal anastomosis. We demonstrate the feasibility of OCT imaging as an intraoperative tool to select conduit vessels for CABG.

Selected historical aspects of the transition of optical coherence tomography (OCT) research from the bench to bedside are focused on. The primary function of the National Institutes of Health (NIH) is to improve the diagnosis and treatment of human pathologies. Therefore, research funded by the NIH should have a direct envisioned pathway for transitioning bench work to the bedside. Ultimately, to be successful, this work must be accepted by physicians and by the general science community. This typically requires robustly validated hypothesis-driven research. Work that is not appropriately compared to the current gold standard or does not address a specific pathology is unlikely to achieve widespread acceptance. I outline OCT research in the musculoskeletal and cardiovascular systems, examining the rapid transition from bench to bedside and look at initial validated hypothesis-driven research data that suggested clinical utility, which drove technology development toward specific clinical scenarios. I also consider the time of initial funding compared to when it was applied in patients with clinical pathologies. Finally, ongoing bench work being performed in parallel with clinical studies is examined. The specific applications examined here are identifying unstable coronary plaque and the early detection of osteoarthritis, the former was brought to the bedside primarily through a commercial route while the latter through NIH-funded research.

Noninvasive optical imaging technology has the potential to improve the accuracy of disease detection and predict treatment response. Pathology provides the critical link between the biological basis of an image or spectral signature and clinical outcomes obtained through optical imaging. The validation of optical images and spectra requires both morphologic diagnosis from histopathology and parametric analysis of tissue features above and beyond the declared pathologic "diagnosis." Enhancement of optical imaging modalities with exogenously applied biomarkers also requires validation of the biological basis for molecular contrast. For an optical diagnostic or prognostic technology to be useful, it must be clinically important, independently informative, and of demonstrated beneficial value to patient care. Its usage must be standardized with regard to methods, interpretation, reproducibility, and reporting, in which the pathologist plays a key role. By providing insight into disease pathobiology, interpretive or quantitative analysis of tissue material, and expertise in molecular diagnosis, the pathologist should be an integral part of any team that is validating novel optical imaging modalities. This review will consider (1) the selection of validation biomarkers; (2) standardization in tissue processing, diagnosis, reporting, and quantitative analysis; (3) the role of the pathologist in study design; and (4) reference standards, controls, and interobserver variability.

Real-time medical imaging systems such as reflectance confocal microscopes and optical coherence microscopes are being tested in multiple-patient and multiple-center clinical trials. The modulation transfer function (MTF) of these systems at any given time influences the image information content and can affect the interpretation of the images. MTF is difficult to measure in real-time scanning systems when imaging at the Nyquist limit. We describe a measurement technique similar to the electronic imaging resolution standards ISO-12233 (electronic cameras) that can be applied to scanned spot imaging systems with asynchronous pixel clocks. This technique requires the acquisition of a single image of a reflective stripe object. An asynchronous pixel clock induces subpixel jitter in the edge location. The jitter is removed using a Fourier method, and an oversampled edge response function is calculated using algorithms developed in MATLAB. This technique provides fast, simple to use, and repeatable full-width at half maximum lateral resolution and MTF measurements based on only one test image. We present the results for reflectance confocal microscopes operating at 0.9 numerical aperature.

Conventional endoscopic images do not provide quantitative 3-D information. We present an endoscope system that can measure the size and position of an object in real time. Our endoscope contains four laser beam sources and a camera. The procedural steps for 3-D measurements are as follows. First, to obtain the function that maps 2-D coordinates of an image point to its 3-D coordinates in 3-D space, we observe a standard chart with the endoscope lens and determine the correspondence between the image and object height. In addition to the mapping, this function can correct barrel-shaped distortion of endoscopic images. The system detects laser spots on an object surface automatically using a template matching method, and maps the 2-D coordinates of the laser spots to the 3-D coordinates by the triangulation method. Then the system calculates the magnification ratio on the object plane, which is perpendicular to the optical axis and passes the laser spot, so that the system can superimpose a ruler whose scale fits the 3-D coordinates of the object. Thus, physicians can measure the size and position of objects in real time on undistorted images similar to placing rulers on the surface of an organ.

We present a method for design and use of a digital mouse phantom for small animal optical imaging. We map the boundary of a mouse model from magnetic resonance imaging (MRI) data through image processing algorithms and discretize the geometry by a finite element (FE) descriptor. We use a validated FE implementation of the three-dimensional (3-D) diffusion equation to model transport of near infrared (NIR) light in the phantom with a mesh resolution optimized for representative tissue optical properties on a computing system with 8-GB RAM. Our simulations demonstrate that a section of the mouse near the light source is adequate for optical system design and that the variation of intensity of light on the boundary is well within typical noise levels for up to 20% variation in optical properties and nodes used to model the boundary of the phantom. We illustrate the use of the phantom in setting goals for specific binding of targeted exogenous fluorescent contrasts based on anatomical location by simulating a nearly tenfold change in the detectability of a 2-mm-deep target depending on its placement. The methodology described is sufficiently general and may be extended to generate digital phantoms for designing clinical optical imaging systems.

We have used quantitative second harmonic generation (SHG) imaging microscopy to investigate the collagen matrix organization in the oim mouse model for human osteogenesis imperfecta (OI). OI is a heritable disease in which the type I collagen fibrils are either abnormally organized or small, resulting in a clinical presentation of recurrent bone fractures and other pathologies related to collagen-comprised tissues. Exploiting the exquisite sensitivity of SHG to supramolecular assembly, we investigated whether this approach can be utilized to differentiate normal and oim tissues. By comparing SHG intensity, fibrillar morphology, polarization anisotropy, and signal directionality, we show that statistically different results are obtained for the wild type (WT) and disease states in bone, tendon, and skin. All these optical signatures are consistent with the collagen matrix in the oim tissues being more disordered, and these results are further consistent with the known weaker mechanical properties of the oim mouse. While the current work shows the ability of SHG to differentiate normal and diseased states in a mouse model, we suggest that our results provide a framework for using SHG as a clinical diagnostic tool for human OI. We further suggest that the SHG metrics described could be applied to other connective tissue disorders that are characterized by abnormal collagen assembly.

Real-time technologies can increase the efficiency of obtaining informative biopsies and accelerate interpretation of biopsy pathological review. Cellular aberrations inherent to cancer cells, including nuclear size, can currently be detected, but few technologies are available to evaluate adequacy of specimens in real time. The aims of this study are: 1. to determine if near-infrared reflectance confocal microscopy (RCM) can be used to assess epithelial/stromal content of core needle breast biopsy samples in real time, 2. to determine if epithelial cell nuclear size can be measured on RCM images, and 3. to test if RCM images can be accurately read for presence/absence of histologically relevant features of malignancy. Breast biopsies are obtained following a medically indicated breast core needle diagnostic biopsy for RCM examination. Acetic acid is used as a contrast agent to visualize structures within breast tissue. Structures are identified and optically serially sectioned, and digital images are cataloged. Relative amounts of epithelial, fatty, and collagenous tissue are determined. RCM biopsies are formalin-fixed and stained for hematoxylin and eosin (H and E) comparison with RCM images. RCM data are comparable to data from H and E sections. Epithelial cell nuclear size is measured on stored digital RCM images. We compare RCM and H and E images from 16 patients and 25 core needle biopsy samples.

We present a novel methodology for combining breast image data obtained at different times, in different geometries, and by different techniques. We combine data based on diffuse optical tomography (DOT) and magnetic resonance imaging (MRI). The software platform integrates advanced multimodal registration and segmentation algorithms, requires minimal user experience, and employs computationally efficient techniques. The resulting superposed 3-D tomographs facilitate tissue analyses based on structural and functional data derived from both modalities, and readily permit enhancement of DOT data reconstruction using MRI-derived a-priori structural information. We demonstrate the multimodal registration method using a simulated phantom, and we present initial patient studies that confirm that tumorous regions in a patient breast found by both imaging modalities exhibit significantly higher total hemoglobin concentration (THC) than surrounding normal tissues. The average THC in the tumorous regions is one to three standard deviations larger than the overall breast average THC for all patients.

We combine diffuse optical spectroscopy (DOS) and diffuse correlation spectroscopy (DCS) to noninvasively monitor early hemodynamic response to neoadjuvant chemotherapy in a breast cancer patient. The potential for early treatment monitoring is demonstrated. Within the first week of treatment (day 7) DOS revealed significant changes in tumor/normal contrast compared to pretreatment (day 0) tissue concentrations of deoxyhemoglobin (rctHHb_T/N=69±21%), oxyhemoglobin (rctO₂Hb_T/N=73±25%), total hemoglobin (rctTHb_T/N=72±17%), and lipid concentration (rctLipid_T/N=116±13%). Similarly, DCS found significant changes in tumor/normal blood flow contrast (rBF_T/N=75±7% on day 7 with respect to day 0). Our observations suggest the combination of DCS and DOS enhances treatment monitoring compared to either technique alone. The hybrid approach also enables construction of indices reflecting tissue metabolic rate of oxygen, which may provide new insights about therapy mechanisms.

Dynamic optical imaging is increasingly applied to clinically relevant areas such as brain and cancer imaging. In this approach, some external stimulus is applied and changes in relevant physiological parameters (e.g., oxy- or deoxyhemoglobin concentrations) are determined. The advantage of this approach is that the prestimulus state can be used as a reference or baseline against which the changes can be calibrated. Here we present the first application of this method to the problem of characterizing joint diseases, especially effects of rheumatoid arthritis (RA) in the proximal interphalangeal finger joints. Using a dual-wavelength tomographic imaging system together with previously implemented model-based iterative image reconstruction schemes, we have performed initial dynamic imaging case studies on a limited number of healthy volunteers and patients diagnosed with RA. Focusing on three cases studies, we illustrated our major finds. These studies support our hypothesis that differences in the vascular reactivity exist between affected and unaffected joints.

Physiological blood coagulation/clotting is an essential biological process that is initiated by vessel injury and includes a cascade of enzymatic reactions finalized by fibrin polymerization and clot formation. We utilize dynamic light scattering (DLS) imaging to monitor in vivo red cell mobility as an indicator of blood coagulation. In the course of the experiments, blood flow is arrested using mechanical occlusion, and then laser injury is applied. We demonstrate that the combination of laser injury with DLS imaging on occluded blood vessels (i.e., under static conditions) is suitable to detect even subtle changes of plasma viscosity in the circulatory system, which reflects the process of clot development. This approach is noninvasive and has a relatively simple and easy-to-use technical design. Thus, the proposed methodology provides a promising tool for investigating blood clotting within the vasculature.

We demonstrate a novel nonintrusive technique based on tunable diode laser absorption spectroscopy to investigate human sinuses in vivo. The technique relies on the fact that free gases have spectral imprints that are about 10.000 times sharper than spectral structures of the surrounding tissue. Two gases are detected; molecular oxygen at 760 nm and water vapor at 935 nm. Light is launched fiber optically into the tissue in close proximity to the particular maxillary sinus under study. When investigating the frontal sinuses, the fiber is positioned onto the caudal part of the frontal bone. Multiply scattered light in both cases is detected externally by a handheld probe. Molecular oxygen is detected in the maxillary sinuses on 11 volunteers, of which one had constantly recurring sinus problems. Significant oxygen absorption imprint differences can be observed between different volunteers and also left-right asymmetries. Water vapor can also be detected, and by normalizing the oxygen signal on the water vapor signal, the sinus oxygen concentration can be assessed. Gas exchange between the sinuses and the nasal cavity is also successfully demonstrated by flushing nitrogen through the nostril. Advantages over current ventilation assessment methods using ionizing radiation are pointed out.

We investigate the feasibility of using diode laser gas spectroscopy for sinusitis diagnostics. We simulate light propagation using the Monte Carlo concept, as implemented by the Advanced Systems Analysis Program (ASAP^TM) software. Simulations and experimental data are compared for a model based on two scattering bodies representing human tissue, with an air gap in-between representing the sinus cavity. Simulations are also performed to investigate the detection geometries used in the experiments, as well as the influence of the optical properties of the scattering bodies. Finally, we explore the possibility of performing imaging measurements of the sinuses. Results suggest that a diagnostic technique complementary to already existing ones could be developed.

We report an analysis of four strains of baker's yeast (Saccharomyces cerevisiae) using biocavity laser spectroscopy. The four strains are grouped in two pairs (wild type and altered), in which one strain differs genetically at a single locus, affecting mitochondrial function. In one pair, the wild-type ρ+ and a ρ0 strain differ by complete removal of mitochondrial DNA (mtDNA). In the second pair, the wild-type ρ+ and a ρ- strain differ by knock-out of the nuclear gene encoding Co×4, an essential subunit of cytochrome c oxidase. The biocavity laser is used to measure the biophysical optic parameter Δλ, a laser wavelength shift relating to the optical density of cell or mitochondria that uniquely reflects its size and biomolecular composition. As such, Δλ is a powerful parameter that rapidly interrogates the biomolecular state of single cells and mitochondria. Wild-type cells and mitochondria produce Gaussian-like distributions with a single peak. In contrast, mutant cells and mitochondria produce leptokurtotic distributions that are asymmetric and highly skewed to the right. These distribution changes could be self-consistently modeled with a single, log-normal distribution undergoing a thousand-fold increase in variance of biomolecular composition. These features reflect a new state of stressed or diseased cells that we call a reactive biomolecular divergence (RBD) that reflects the vital interdependence of mitochondria and the nucleus.

We develop a new approach in imaging nonfluorescent species with two-color two-photon and excited state absorption microscopy. If one of two synchronized mode-locked pulse trains at different colors is intensity modulated, the modulation transfers to the other pulse train when nonlinear absorption takes places in the medium. We can easily measure 10^-6 absorption changes caused by either two-photon absorption or excited-state absorption with a RF lock-in amplifier. Sepia melanin is studied in detail as a model system. Spectroscopy studies on the instantaneous two-photon absorption (TPA) and the relatively long-lived excited-state absorption (ESA) of melanin are carried out in solution, and imaging capability is demonstrated in B16 cells. It is found that sepia melanin exhibits two distinct excited states with different lifetimes (one at 3 ps, one lasting hundreds of nanoseconds) when pumped at 775 nm. Its characteristic TPA/ESA enables us to image its distribution in cell samples with high resolution comparable to two-photon fluorescence microscopy (TPFM). This new technique could potentially provide valuable information in diagnosing melanoma.

The intrinsic optical parameters—absorption coefficient µa, scattering coefficient μ_a, anisotropy factor g, and effective scattering coefficient μ^′_s—are determined for human red blood cells of hematocrit 42.1% dependent on the shear rate in the wavelength range 250 to 1100 nm. Integrating sphere measurements of light transmittance and reflectance in combination with inverse Monte-Carlo simulation are carried out for different wall shear rates between 0 and 1000 s^-1. Randomly oriented cells show maximal μ_a, μ_s, and μ^′_s values. Cell alignment and elongation, as well as the Fahraeus effect at increasing shear rates, lead to an asymptotical decrease of these values. The anisotropy factor shows this behavior only below 600 nm, dependent on absorption; above 600 nm, g is almost independent of shear rate. The decrease of μ^′_s is inversely correlated with the hemoglobin absorption. Compared to randomly oriented cells, aggregation reduces all parameters by a different degree, depending on the hemoglobin absorption. It is possible to evaluate the influence of collective scattering phenomena, the absorption within the cell, and the cell shape.

A method is proposed for visualizing the depth and thickness distribution of a local blood region in skin tissue using diffuse reflectance images at three isosbestic wavelengths of hemoglobin: 420, 585, and 800 nm. Monte Carlo simulation of light transport specifies a relation among optical densities, depth, and thickness of the region under given concentrations of melanin in epidermis and blood in dermis. Experiments with tissue-like agar gel phantoms indicate that a simple circular blood region embedded in scattering media can be visualized with errors of 6% for the depth and 22% for the thickness to the given values. In-vivo measurements on human veins demonstrate that results from the proposed method agree within errors of 30 and 19% for the depth and thickness, respectively, with values obtained from the same veins by the conventional ultrasound technique. Numerical investigation with the Monte Carlo simulation of light transport in the skin tissue is also performed to discuss effects of deviation in scattering coefficients of skin tissue and absorption coefficients of the local blood region from the typical values of the results. The depth of the local blood region is over- or underestimated as the scattering coefficients of epidermis and dermis decrease or increase, respectively, while the thickness of the region agrees well with the given values below 1.2 mm. Decreases or increases of hematocrit value give over- or underestimation of the thickness, but they have almost no influence on the depth.

Arterial tissues collected from Ossabaw swine bearing metabolic syndrome-induced cardiovascular plaques are characterized by multimodal nonlinear optical microscopy that allows coherent anti-Stokes Raman scattering, second-harmonic generation, and two-photon excitation fluorescence imaging on the same platform. Significant components of arterial walls and atherosclerotic lesions, including endothelial cells, extracellular lipid droplets, lipid-rich cells, low-density lipoprotein aggregates, collagen, and elastin are imaged without any labeling. Emission spectra of these components are obtained by nonlinear optical microspectrometry. The nonlinear optical contrast is compared with histology of the same sample. Multimodal nonlinear optical imaging of plaque composition also allows identification of atherosclerotic regions that are vulnerable to rupture risk. The demonstrated capability of nonlinear optical microscopy for label-free molecular imaging of atherosclerotic lesions with 3-D submicrometric resolution suggests its potential application to the diagnosis of atherosclerotic plaques, determination of their rupture risk, and design of individualized drug therapy based on plaque composition.

Structural proteins such as elastin and collagen can be readily imaged by using two-photon excitation and second-harmonic generation microscopic techniques, respectively, without physical or biochemical processing of the tissues. This time- and effort-saving advantage makes these imaging techniques convenient for determining the structural characteristics of blood vessels in vivo. Fibrillar collagen is a well-known element involved in the formation of atherosclerotic lesions. It is also an important component of the fibrous cap responsible for structural stability of atherosclerotic plaques. High resolution in vivo microscopic imaging and characterization of atherosclerotic lesions in animal models can be particularly useful for drug discovery. However, it is hindered by the limitations of regular microscope objectives to gain access of the tissues of interest and motional artifacts. We report a technique that facilitates in vivo microscopic imaging of carotid arteries of rodents using conventional microscope objectives, and at the same time avoids motional artifacts. As a result, collagen, elastin, leukocytes, cell nuclei, and neutral lipids can be visualized in three dimensions in live animals. We present and discuss in vivo imaging results using a flow cessation mouse model of accelerated atherosclerosis.

A method for the determination of the integral refractive index of living cells in suspension by digital holographic microscopy is described. Digital holographic phase contrast images of spherical cells in suspension are recorded, and the radius as well as the integral refractive index are determined by fitting the relation between cell thickness and phase distribution to the measured phase data. The algorithm only requires information about the refractive index of the suspension medium and the image scale of the microscope system. The specific digital holographic microscopy advantage of subsequent focus correction allows a simultaneous investigation of cells in different focus planes. Results obtained from human pancreas and liver tumor cells show that the integral cellular refractive index decreases with increasing cell radius.

Noninvasive measurement of 3-D morphology of adhered animal cells employing a phase-shifting laser microscope (PLM) is investigated, in which the phase shift for each pixel in the view field caused by cell height and the difference in refractive indices between the cells and the medium is determined. By employing saline with different refractive indices instead of a culture medium, the refractive index of the cells, which is necessary for the determination of cell height, is determined under PLM. The observed height of Chinese hamster ovary (CHO) cells cultivated under higher osmolarity is lower than that of the cells cultivated under physiological osmolarity, which is in agreement with previous data observed under an atomic force microscope (AFM). Maximum heights of human bone marrow mesenchymal stem cells and human umbilical cord vein endothelial cells measured under PLM and AFM agree well with each other. The maximum height of nonadherent spherical CHO cells observed under PLM is comparable to the cell diameter measured under a phase contrast inverted microscope. Laser irradiation, which is necessary for the observation under PLM, did not affect 3-D cell morphology. In conclusion, 3-D morphology of adhered animal cells can be noninvasively measured under PLM.

We have previously demonstrated that Förster resonance energy transfer (FRET) efficiency and the relative concentration of donor and acceptor fluorophores can be determined in living cells using three-cube wide-field fluorescence microscopy. Here, we extend the methodology to estimate the effective equilibrium dissociation constant (K_d) and the intrinsic FRET efficiency (E_max) of an interacting donor-acceptor pair. Assuming bimolecular interaction, the predicted FRET efficiency is a function of donor concentration, acceptor concentration, K_d, and E_max. We estimate K_d and E_max by minimizing the sum of the squared error (SSE) between the predicted and measured FRET efficiency. This is accomplished by examining the topology of SSE values for a matrix of hypothetical K_d and E_max values. Applying an F-test, the 95% confidence contour of K_d and E_max is calculated. We test the method by expressing an inducible FRET fusion pair consisting of FKBP12-Cerulean and Frb-Venus in HeLa cells. As the K_d for FKBP12-rapamycin and Frb has been analytically determined, the relative Kd (in fluorescence units) could be calibrated with a value based on protein concentration. The described methodology should be useful for comparing protein-protein interaction affinities in living cells.

Studies of bioluminescence in living animals, such as cell-based biosensor applications, require measurement of light at different wavelengths, but accurate light measurement is impeded by absorption by tissues at wavelengths <600 nm. We present a novel approach to this problem—the use of a plastic window in the skin/body wall of mice—that permits measurements of light produced by bioluminescent cells transplanted into the kidney. The cells coexpressed firefly luciferase (FLuc), a vasopressin receptor—Renilla luciferase (RLuc) fusion protein, and a GFP²-β-arrestin2 fusion protein. Following coadministration of two luciferase substrates, native coelenterazine and luciferin, bioluminescence is measured via the window using fiber optics and a photon counter. Light emission from the two different luciferases, FLuc and RLuc, is readily distinguishable using appropriate optical filters. When coelenterazine 400a is administered, bioluminescence resonance energy transfer (BRET) occurs between the RLuc and GFP² fusion proteins and is detected by the use of suitable filters. Following intraperitoneal injection of vasopressin, there is a marked increase in BRET. When rapid and accurate measurement of light from internal organs is required, rather than spatial imaging of bioluminescence, the combination of skin/body wall window and fiber optic light measurement will be advantageous.

Receiver operating characteristic (ROC) analysis was performed on simulated near-infrared tomography images, using both human observer and contrast-to-noise ratio (CNR) computational assessment, for application in breast cancer imaging. In the analysis, a nonparametric approach was applied for estimating the ROC curves. Human observer detection of objects had superior capability to localize the presence of heterogeneities when the objects were small with high contrast, with a minimum detectable threshold of CNR near 3.0 to 3.3 in the images. Human observers were able to detect heterogeneities in the images below a size limit of 4 mm, yet could not accurately find the location of these objects when they were below 10 mm diameter. For large objects, the lower limit of a detectable contrast limit was near 10% increase relative to the background. The results also indicate that iterations of the nonlinear reconstruction algorithm beyond 4 did not significantly improve the human detection ability, and degraded the overall localization ability for the objects in the image, predominantly by increasing the noise in the background. Interobserver variance performance in detecting objects in these images was low, suggesting that because of the low spatial resolution, detection tasks with NIR tomography is likely consistent between human observers.

Near-infrared optical imaging is an emerging noninvasive technology toward breast cancer diagnosis. The optical imaging systems available to date are limited either by flexibility to image any given breast volume, patient comfort, or instrument portability. Here, a hand-held optical probe is designed and developed, 1. employing a unique measurement scheme of simultaneous multiple point illumination and collection for rapid data acquisition and minimal patient discomfort, and 2. employing a curved probe head such that it allows flexible imaging of tissue curvatures. Simulation studies are carried out on homogeneous slab phantoms (5×10×8 cc) to determine an appropriate source-detector configuration for the probe head. These design features are implemented in the development of the probe, which consisted of six simultaneous illuminating and 165 simultaneous collecting fibers, spaced 0.5 cm apart on a 5×10 sq-cm probe head. Simulation studies on 3-D slab and curved phantoms demonstrate an increase in the total area of predicted fluorescence amplitude and overall signal strength on using simultaneous multiple point sources over a single point source. The probe is designed and developed such that on coupling with a detection system in the future, the hand-held probe based imager can be clinically assessed toward cancer diagnostic imaging.

Surface-enhanced Raman scattering (SERS) is a powerful technique for the analysis of a variety of molecules and molecular structures. Due to its great complexity, the acquisition of detailed molecular information from biological organizations such as bacteria is still a challenging task. SERS can provide valuable information once silver or gold surfaces can be brought in close contact with the biological organization. Because several experimental parameters can affect SERS spectra of bacteria, the experimental conditions must be well defined for comparable and reproducible results. The influence of experimental parameters, such as the type of noble metal, size, and aggregation properties of nanoparticles, and the wavelength of the laser light on the SERS of E. coli and B. megaterium are examined. It is demonstrated that the impact of these parameters could be enormous and a standard protocol must be developed depending on the goal of the study.

Separation and transport of defined populations of living cells grown on a thin membrane can be achieved by laser microdissection (LMD) of the sample of interest, followed by a laser-induced forward transport process [laser pressure "catapulting" (LPC)] of the dissected cell cluster. We investigate the dynamics of LMD and LPC with focused and defocused UV-A laser pulses by means of time-resolved photography. Catapulting is driven by plasma formation when tightly focused pulses are used, and by confined thermal ablation at the bottom of the sample for defocused catapulting. With both modalities, the initial specimen velocity amounts to about 50 to 60 m/s. Time-resolved photography of live cell catapulting reveals that in defocused catapulting, strong shear forces arise when the sample is accelerated out of the culture medium covering the cells. By contrast, pulses focused at the periphery of the specimen cause a fast rotational movement that minimizes the flow of culture medium parallel to the sample surface, and thus the resulting shear stresses. Therefore, the recultivation rate of catapulted cells is much higher when focused pulses are used. Compared to collateral damage by mechanical forces, side effects by heat and UV exposure of the cells play only a minor role.

A passive, optical cell sorter is created using the light pattern of a 'nondiffracting' beam—the Bessel beam. As a precursor to cell sorting studies, microspheres are used to test the resolution of the sorter on the basis of particle size and refractive index. Variations in size and, more noticeably, refractive index, lead to a marked difference in the migration time of spheres in the Bessel beam. Intrinsic differences (size, refractive index) between native (unlabeled) cell populations are utilized for cell sorting. The large difference in size between erythrocytes and lymphocytes results in their successful separation in this beam pattern. The intrinsic differences in size and refractive index of other cells in the study (HL60 human promyelocytic leukaemic cells, murine bone marrow, and murine stem/progenitor cells) are not large enough to induce passive optical separation. Silica microsphere tags are attached to cells of interest to modify their size and refractive index, resulting in the separation of labeled cells. Cells collected after separation are viable, as evidenced by trypan blue dye exclusion, their ability to clone in vitro, continued growth in culture, and lack of expression of Caspase 3, a marker of apoptosis.

For bioluminescence imaging (BLI) of small animals, the most commonly used luciferase is Fluc from the firefly, but recently, green (CBGr99) and red (CBRed) click beetle luciferases became available. Because signal attenuation by tissues is lower for red light, red luciferases appear to be advantageous for BLI, but this has not been thoroughly tested. We compare different luciferases for BLI. For this purpose, cell transfectants are generated expressing comparable amounts of CBGr99, CBRed, or Fluc. This is achieved by coexpression of the luciferase with eGFP using the bicistronic 2A system, which results in stoichiometric coexpression of the respective proteins. In vitro, the CBGr99 transfectant exhibits the strongest total photon yield. For in vivo BLI, the transfectants are injected into mice at different locations. At a subcutaneous position, CBGr99 is clearly superior to the other luciferases. When the tumor cells are located in the peritoneum or lung, where more absorption by tissue occurs, CBGr99 and CBRed transfected cells emit a comparable number of red photons and are superior to Fluc, but CBGr99 reaches the maximum of the light emission faster than CBRed. Thus, although CBGr99 emits mainly green light, the high yield of total and red photons makes it an excellent candidate for BLI.

We developed a novel scheme for two-photon fluorescence bioimaging. We generated supercontinuum (SC) light at wavelengths of 600 to 1200 nm with 774-nm light pulses from a compact turn-key semiconductor laser picosecond light pulse source that we developed. The supercontinuum light was sliced at around 1030- and 920-nm wavelengths and was amplified to kW-peak-power level using laboratory-made low-nonlinear-effects optical fiber amplifiers. We successfully demonstrated two-photon fluorescence bioimaging of mouse brain neurons containing green fluorescent protein (GFP).

This PDF file contains the errata for “JBO Vol. 12 Issue 05 Paper 2803891” for JBO Vol. 12 Issue 05