Confocal laser scanning microscopy of retinal rod outer segment intact disks: new labeling techniqueRavera, Silvia
2007 Journal of Biomedical Optics
doi: 10.1117/1.2790919pmid: 17994858
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.
In vivo multiphoton nanosurgery on cortical neuronsSacconi, Leonardo
2007 Journal of Biomedical Optics
doi: 10.1117/1.2798723pmid: 17994859
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.
Subcellular imaging of epithelium with time-lapse optical coherence tomographyPan, Ying T.
2007 Journal of Biomedical Optics
doi: 10.1117/1.2800007pmid: 17994860
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 ॖ m ) closely matches the histological evaluation (7.2±0.8 ॖ m ) . 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 .
Detection and treatment of dysplasia in Barrett’s esophagus: a pivotal challenge in translating biophotonics from bench to bedsideWilson, Brian C.
2007 Journal of Biomedical Optics
doi: 10.1117/1.2795688pmid: 17994862
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 in vivo : techniques and applications from animal to manHillman, Elizabeth M. C.
2007 Journal of Biomedical Optics
doi: 10.1117/1.2789693pmid: 17994863
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.
Optical coherence tomography: a review of clinical development from bench to bedsideZysk, Adam M.; Nguyen, Freddy T.; Oldenburg, Amy L.; Boppart, Stephen A.
2007 Journal of Biomedical Optics
doi: 10.1117/1.2793736pmid: 17994864
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.
D-galactose receptor-targeted in vivo spectral fluorescence imaging of peritoneal metastasis using galactosamin-conjugated serum albumin-rhodamine greenHama, Yukihiro; Urano, Yasuteru; Koyama, Yoshinori
2007 Journal of Biomedical Optics
doi: 10.1117/1.2779351pmid: 17994865
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&percent;/99&percent; (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.
Imaging Probe Development Center: a National Institutes of Health core synthesis resource for imaging probesShi, Zhen-Dan
2007 Journal of Biomedical Optics
doi: 10.1117/1.2778702pmid: 17994866
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.
Photoacoustic flow cytometry: principle and application for real-time detection of circulating single nanoparticles, pathogens, and contrast dyes in vivoZharov, Vladimir P.; Galanzha, Ekaterina I.; Shashkov, Evgeny V.; Kim, Jin-Woo; Khlebtsov, Nikolai G.; Tuchin, Valery V.
2007 Journal of Biomedical Optics
doi: 10.1117/1.2793746pmid: 17994867
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 10 8 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.