KEYWORDS: Luminescence, High dynamic range imaging, Signal to noise ratio, Range imaging, Microscopy, Image fusion, Image quality, Sensors, Polarization
Significance: Fluorescence polarization (FP) and fluorescence anisotropy (FA) microscopy are powerful imaging techniques that allow to translate the common FP assay capabilities into the in vitro and in vivo cellular domain. As a result, they have found potential for mapping drug–protein or protein–protein interactions. Unfortunately, these imaging modalities are ratiometric in nature and as such they suffer from excessive noise even under regular imaging conditions, preventing accurate image-feature analysis of fluorescent molecules behaviors.
Aim: We present a high dynamic range (HDR)-based FA imaging modality for improving image quality in FA microscopy.
Approach: The method exploits ad hoc acquisition schemes to extend the dynamic range of individual FP channels, allowing to obtain FA images with increased signal-to-noise ratio.
Results: A direct comparison between FA images obtained with our method and the standard, clearly indicates how an HDR-based FA imaging approach allows to obtain high-quality images, with the ability to correctly resolve image features at different values of FA and over a substantially higher range of fluorescence intensities.
Conclusion: The method presented is shown to outperform standard FA imaging microscopy narrowing the spread of the propagated error and yielding higher quality images. The method can be effectively and routinely used on any commercial imaging system and could be also translated to other microscopy ratiometric imaging modalities.
Respiratory- and cardiac-induced motion artifacts pose a major challenge for in vivo optical imaging, limiting the temporal and spatial imaging resolution in fluorescence laser scanning microscopy. Here, we present an imaging platform developed for in vivo characterization of physiologically induced axial motion. The motion characterization system can be straightforwardly implemented on any conventional laser scanning microscope and can be used to evaluate the effectiveness of different motion stabilization schemes. This method is particularly useful to improve the design of novel tissue stabilizers and to facilitate stabilizer positioning in real time, therefore facilitating optimal tissue immobilization and minimizing motion induced artifacts.
A major challenge in high-resolution intravital confocal and multiphoton microscopy is physiologic tissue movement during image acquisition. Of the various physiological sources of movement, respiration has arguably the largest and most wide-ranging effect. We describe a technique for achieving stabilized microscopy imaging using a dual strategy. First, we designed a mechanical stabilizer for constraining physical motion; this served to simultaneously increase the in-focus range over which data can be acquired as well as increase the reproducibility of imaging a certain position within each confocal imaging plane. Second, by implementing a retrospective breathing-gated imaging modality, we performed selective image extraction gated to a particular phase of the respiratory cycle. Thanks to the high reproducibility in position, all gated images presented a high degree of correlation over time. The images obtained using this technique not only showed significant improvements over images acquired without the stabilizer, but also demonstrated accurate in vivo imaging during longitudinal studies. The described methodology is easy to implement with any commercial imaging system, as are used by most biological imaging laboratories, and can be used for both confocal and multiphoton laser scanning microscopy.
KEYWORDS: Luminescence, Intravascular ultrasound, Imaging systems, In vivo imaging, Algorithm development, Signal attenuation, Arteries, Blood, Biology, Near infrared
Intravascular Near-Infrared Fluorescence (NIRF) imaging is a promising imaging modality to image vessel
biology and high-risk plaques in vivo. We have developed a NIRF fiber optic catheter and have presented
the ability to image atherosclerotic plaques in vivo, using appropriate NIR fluorescent probes. Our
catheter consists of a 100/140 μm core/clad diameter housed in polyethylene tubing, emitting NIR laser
light at a 90 degree angle compared to the fiber's axis. The system utilizes a rotational and a
translational motor for true 2D imaging and operates in conjunction with a coaxial intravascular
ultrasound (IVUS) device. IVUS datasets provide 3D images of the internal structure of arteries and are
used in our system for anatomical mapping. Using the IVUS images, we are building an accurate hybrid
fluorescence-IVUS data inversion scheme that takes into account photon propagation through the blood
filled lumen. This hybrid imaging approach can then correct for the non-linear dependence of light
intensity on the distance of the fluorescence region from the fiber tip, leading to quantitative imaging.
The experimental and algorithmic developments will be presented and the effectiveness of the
algorithm showcased with experimental results in both saline and blood-like preparations. The
combined structural and molecular information obtained from these two imaging modalities are
positioned to enable the accurate diagnosis of biologically high-risk atherosclerotic plaques in the
coronary arteries that are responsible for heart attacks.
In this work, we have developed a selective-plane illumination multispectral optoacoustic tomography (MSOT)
technique for high-resolution whole-body visualization of intact optically diffusive organisms whose sizes may vary
from sub-millimeter up to a centimeter range and beyond. By combining multi-wavelength illumination, the method is
shown capable of resolving tissue-specific expression of fluorescent proteins and other molecular biomarkers located
deep in living optically diffuse tissues.
KEYWORDS: Luminescence, Molecular imaging, In vivo imaging, Blood, Signal detection, Near infrared, Inflammation, Imaging systems, Arteries, Visualization
New imaging methods are urgently needed to identify high-risk atherosclerotic lesions prior to the onset of myocardial infarction, stroke, and ischemic limbs. Molecular imaging offers a new approach to visualize key biological features that characterize high-risk plaques associated with cardiovascular events. While substantial progress has been realized in clinical molecular imaging of plaques in larger arterial vessels (carotid, aorta, iliac), there remains a compelling, unmet need to develop molecular imaging strategies targeted to high-risk plaques in human coronary arteries. We present recent developments in intravascular near-IR fluorescence catheter-based strategies for in vivo detection of plaque inflammation in coronary-sized arteries. In particular, the biological, light transmission, imaging agent, and engineering principles that underlie a new intravascular near-IR fluorescence sensing method are discussed. Intravascular near-IR fluorescence catheters appear highly translatable to the cardiac catheterization laboratory, and thus may offer a new in vivo method to detect high-risk coronary plaques and to assess novel atherosclerosis biologics.
The ability to image polarization-selective tissue structures may provide valuable information on tissue anatomy,
morphogenesis, and disease progression. So far, intensive light scattering in biological medium has limited
implementation of polarization imaging to superficial tissue layers. We suggest overcoming the scattering problem using
polarization-sensitive optoacoustic imaging. Due to intrinsically high spatial resolution and sensitivity of the method, it
holds promise of becoming highly accurate modality for interrogation of small polarized structures deep in biological
tissues. We show initial tomographic results in tissue-mimicking phantoms having polarization dichroism contrast.
Noninvasive imaging of biological tissues using visible and near-infrared light may provide numerous insights into
the underlying morphology or tissue function using a great variety of contrast and probing mechanisms. Nevertheless,
mesoscopic-scale (i.e 1mm-1cm sized) living organisms remain largely inaccessible by current optical imaging methods.
Depending on the optical properties of a particular object, light diffusion can significantly limit the resolution that can be
achieved at depths beyond several hundred microns. To enable in-vivo optical contrast imaging of many important
model organisms, such as insects, worms and similarly sized biological specimens, we have developed a multi-spectral
optoacoustic tomography technique for high-resolution imaging of optically diffusive organisms and tissues. The method
is capable of imaging at depths from sub-millimeter up to a centimeter range with a scalable spatial resolution on the
order of magnitude of a few tenths of microns. Furthermore, we show for the first time that the technique is capable of
resolving spatial distribution of fluorescent proteins inside intact opaque organisms, thus overcoming depth limitations of
current fluorescence microscopy techniques.
Current non-invasive imaging methods of fluorescent molecular probes in the visible and near-infrared suffer from low
spatial resolution as a result of rapid light diffusion in biological tissues. We show that three-dimensional distribution of
fluorochromes deep in small animals can be resolved with below 25 femtomole sensitivity and 150 microns spatial
resolution by means of multi-spectral photoacoustic molecular tomography. The low sensitivity limit of the method is
enabled by using the highly resonant absorption spectrum of a commonly used near-infrared fluorescent molecular probe
Alexa Fluor® 750 in order to acquire differential images at multiple wavelengths with tomographic topology suitable for
whole-body small animal imaging.
We have constructed an integrated optical microscope that allows for the simultaneous image acquisition from multiple optical imaging modalities. The microscope consists primarily of hardware for spectral-domain optical coherence microscopy (OCM), multi-photon microscopy (MPM), and second harmonic generation microscopy. The unique configuration of the integrated microscope allows for the acquisition of both anatomical and functional imaging information with particular emphasis in the fields of tissue engineering and tumor biology. By overlaying the structural image obtained from OCM on the functional image obtained simultaneously from MPM (i.e., fluorescent markers imply functional proteins), a more comprehensive view of different tissues can be obtained. In addition, the contemporary analysis of the spectroscopic features enhances contrast in OCM by differentiating different cell and tissue components.
Cell-based engineered tissue models have been increasingly useful in the field of tissue engineering, in in vitro drug screening systems, and in complex cell biology studies. While techniques for engineering tissue models have advanced, there have been few imaging technique capable of assessing the complex 3-D cell behaviors in real-time and at the depths that comprise thick tissues. Understanding cell behavior requires advanced imaging tools to progress from characterizing 2-D cell cultures to complex, highly-scattering, thick 3-D tissue constructs. In this study, we demonstrate that it is possible to use OCT to non-destructively evaluate dynamic cell behavior and function in a quantitative fashion in four dimensions (3-D space plus time). Dynamic processes including cell migration, proliferation, apoptosis, necrosis, and mechanical restructuring are observed during engineering tissue development. With high penetration depth and increased spatial and temporal resolution in 3-D space, OCT will be a useful tool for improving our understanding of cell dynamics in situ and in real-time, for elucidating the complex biological interactions, and for directing our designs toward functional and biomimetic engineered tissues.
We explore combining Coherent anti-Stokes Raman Scattering
with Optical Coherence Tomography ranging when low numerical aperture scanning is used, and demonstrate ranging of layers in a Raman-active medium.
Diagnostic imaging trends are progressing toward the molecular level with the advent of molecular imaging techniques. Optical molecular imaging techniques that utilize targeted exogenous contrast agents or detect endogenous molecular signatures will significantly extend our ability to detect early pathological changes in biological tissue, and treat diseases early when they are most amenable to be cured. We have developed a technique called Nonlinear Interferometric Vibrational Imaging (NIVI) that takes advantage of the coherent nature of coherent anti-Stokes Raman scattering (CARS) processes and the coherent optical ranging and imaging capabilities of optical coherence tomography (OCT). OCT uses interferometry and heterodyne detection in the time or spectral domain to localize reflections of near-infrared light deep from within highly-scattering tissues. OCT has found wide biological and medical applications, and recently, molecular imaging methods have been developed. By utilizing the molecular-sensitivity of CARS, NIVI performs optical ranging and multi-dimensional molecular imaging with OCT-like optical systems, enabling the retrieval of not only χ(3) [chi(3)] amplitude but also phase information, the rejection of problematic non-resonant background four-wave-mixing signals, enhanced sensitivity from heterodyne detection, and a relaxation of the high-numerical aperture focusing requirements present in CARS microscopy. We present recent progress and advances in NIVI, including depth-ranging capabilities that extend significantly deeper than current CARS microscopy methods and are potentially more suitable for cross-sectional deep-tissue in vivo imaging.
Vibrationally-sensitive spectroscopic techniques are becoming important clinical tools for real-time, in vivo diagnostics. The molecular information made available with these techniques can provide early diagnostic signs of disease, often before morphological changes occur. We model and experimentally demonstrate a new technique for measuring optical spectroscopy signals using interferometric ranging. This new technique, nonlinear interferometric vibrational imaging (NIVI), uses principles from coherent anti-Stokes Raman scattering (CARS) spectroscopy and optical coherence tomography (OCT) to achieve cross-sectional imaging of the distribution of specific molecular species within a sample. Two CARS signals are generated, one from a known reference molecular species and a second from the unknown molecules in a sample. These coherent signals are interfered with each other using an interferometer setup. The intensity envelope of the interference signal provides a measure of the concentration of selected bonds present in the sample focal volume. The interference fringes themselves can provide phase information that will allow for the exact reconstruction of the vibrational characteristics of the molecules in the sample focal volume. Theoretical background to CARS interferometry is presented, the experimental laser systems are described, and a depth-resolved scan line of a benzene filled cuvette is demonstrated. The experimental results show close resemblance to the theoretical models. The advantages of NIVI over existing vibrational imaging systems and its clinical implications are discussed.
Recent advances in the field of tissue engineering have led to the development of complex three-dimensional tissue constructs. It has become clear, however, that the traditional tools used for studying standard cell cultures are not always adequate for diagnostically studying thick, highly-scattering cultured tissues. Furthermore, many techniques used for studying three-dimensional constructs are invasive or require exogenous fluorophores, which damage the tissue and prevent time-course studies of tissue development. An integrated optical coherence tomography (OCT) and multi-photon microscope (MPM) has been constructed for visualizing 3-D engineered tissues. OCT was used for imaging structure and cell organization, while MPM was used for assessing functional properties of cells. We demonstrate technical developments involved in the construction of this instrument and its use in the non-destructive investigation of cell movement and tissue organization in engineered tissues. Cells labeled with GFP and exogenous fluorescent probes have also been imaged with OCT and confocal microscopy. Studies indicate that an integrated microscope has the potential to be an enabling diagnostic tool for future studies in the growth and organization of engineering tissues and in cell-cell and cell-matrix interactions.
We report PMD effect on the distributed chromatic dispersion map measurement based on phase-mismatched four-wave mixing. The experimental results of distributed chromatic dispersion maps for a low-PMD dispersion-shifted fiber are described with spatial resolution of 250 m and dispersion accuracy less than ±0.02 ps/nm.km. For high-PMD DSF fibers, the chromatic dispersion map may be difficult to determine if the fiber has a long polarization-coupling length; however, it is still possible to be measured for a low polarization-coupling length fiber. Finally, the use of this method to determine distributed nonlinear coefficient will also be discussed.
We present a systematic study of the growth of polycrystalline diamond thin films on W wires and tips by hot filament assisted chemical vapor deposition for x-ray detection purposes. We carry out correlations between scanning electron microscopy (SEM) observations and micro- Raman spectra, while varying different growth parameters. SEM observations show a uniform covering of the substrate, with growth rates ranging from 0.5 to 1.5 micrometers /h. All (mu) - R spectra show a well defined diamond peak at 1330.8-1333.7 cm-1 together with abroad structure at 1400-1600 cm-1 and a luminescence background extending over the whole scanned range. A close analysis shows that best quality is obtained with the lowest diameter substrates, at the lowest CH4 concentration and at a low pressure. Some depositions have been studied as x-ray detectors and their sensitivity at low energy and 6 MeV beam evaluated, showing a good response with respect to standard ionization chambers.
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