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A molecular laser-radar system, based on the Scheimpflug principle, has been constructed and demonstrated for remote sensing of atmospheric CO

A novel pathlength enhancement approach has been applied in multimode diode laser correlation spectroscopy measurements of gas concentrations. Reference and sample gas cells made of porous polystyrene foam and alumina ceramic, respectively, were employed in proof-of-principle measurements of molecular oxygen. Equivalent pathlengths of 164 and 52 cm were obtained for the reference and sample cells with physical pathlengths of 4 and 1 cm, respectively. With a measurement time of 60 s, a physical-pathlength-integrated sensitivity of 15 ppm⋅m was achieved with an enhancement of more than one order of magnitude compared with an open-air setup. Practical application aspects were investigated including gas cell size and light alignment tolerance. This generic approach for pathlength enhancement provides advantages in terms of compactness and robustness.

Fluorescence diffuse optical tomography (FDOT) is an emerging biomedical imaging technique that can be used to localize and quantify deeply situated fluorescent molecules within tissues. However, the potential of this technique is currently limited by its poor spatial resolution. In this work, we demonstrate that the current resolution limit of FDOT can be breached by exploiting the nonlinear power-dependent optical emission property of upconverting nanoparticles doped with rare-earth elements. The rare-earth-doped core-shell nanoparticles, NaYF(4):Yb(3+)/Tm(3+)@NaYF(4) of hexagonal phase, are synthesized through a stoichiometric method, and optical characterization shows that the upconverting emission of the nanoparticles in tissues depends quadratically on the power of excitation. In addition, quantum-yield measurements of the emission from the synthesized nanoparticles are performed over a large range of excitation intensities, for both core and core-shell particles. The measurements show that the quantum yield of the 800 nm emission band of core-shell upconverting nanoparticles is 3.5% under an excitation intensity of 78 W/cm(2). The FDOT reconstruction experiments are carried out in a controlled environment using liquid tissue phantoms. The experiments show that the spatial resolution of the FDOT reconstruction images can be significantly improved by the use of the synthesized upconverting nanoparticles and break the current spatial resolution limits of FDOT images obtained from using conventional linear fluorophores as contrast agents.

Purpose: To study the feasibility of using transscleral visible/near-infrared spectroscopy (Vis/NIRS) to estimate the content of haemoglobin in choroidal tumour phantoms of ex vivo porcine eyes. Methods: Thirty enucleated porcine eyes were prepared with a tumour phantom made by injecting a suspension of gelatine, titanium dioxide and human blood into the suprachoroidal space. The blood concentrations used were 2.5%, 25% and 50%, with 10 eyes in each group. Alternating Vis/NIRS measurements were taken over the phantom inclusion and on the opposite (normal) side of each eye. For statistical analysis, a genetic algorithm was utilized to suppress insignificant wavelengths in the spectra. The processed spectra were then used to build a regression model based on partial least squares regression and evaluated by twofold cross-validation. Results: Ultrasonography revealed that all phantoms were localized within the suprachoroidal space with no penetration through the retina. The largest mean diameters of the phantoms with 2.5%, 25% and 50% blood were 15.5, 15.2 and 15.7 mm, respectively (p > 0.05). The largest mean thicknesses were 4.5, 4.5 and 4.8 mm, respectively (p > 0.05). Statistical analysis of the spectral data showed that it was possible to correctly discriminate between the normal side and the tumour phantom side of the eyes in 99.88% of cases. The phantoms could be correctly classified according to their blood concentrations in 99.42% of cases. Conclusions: This study demonstrates that transscleral Vis/NIRS is a feasible and accurate method for the detection of choroidal tumours and to assess the haemoglobin content in such lesions. (Less)

The spatial resolution of fluorescence molecular imaging is a critical issue for the success of the technique in biomedical applications. One important method for increasing the imaging resolution is to utilize multi-photon emissions. In this study, we thoroughly investigate the potential of the multi-photon upconversion emissions from rare-earth-doped upconverting nanoparticles for the improvement in spatial resolution of diffuse optical imaging. It is found that the imaging resolution is increased by a factor of 1.45 through employing two-photon upconversion emission compared with using the linear emission, and can be further elevated by a factor of 1.23 by using three-photon upconversion emission. In addition, we demonstrate that the pulsed excitation approach holds the promise of overcoming the low quantum yield associated with the high-order upconversion emissions.

Upconverting nanoparticles suffer from low quantum yield in diffuse optical imaging, especially at low excitation intensities. Here, the power density dependent quantum yield is characterized, and the excitation scheme is optimized based on such characterization

Upconverting nanoparticles (UCNPs) have recently shown great potential as contrast agents in biological applications. In developing different UCNPs, the characterization of their quantum yield (QY) is a crucial issue, as the typically drastic decrease in QY for low excitation power densities can either impose a severe limitation or provide an opportunity in many applications. The power density dependence of the QY is governed by the competition between the energy transfer upconversion (ETU) rate and the linear decay rate in the depopulation of the intermediate state of the involved activator in the upconversion process. Here we show that the QYs of Yb(3+) sensitized two-photon upconversion emissions can be well characterized by the balancing power density, at which the ETU rate and the linear decay rate have equal contributions, and its corresponding QY. The results in this paper provide a method to fully describe the QY of upconverting nanoparticles for arbitrary excitation power densities, and is a fast and simple approach for assessing the applicability of UCNPs from the perspective of energy conversion.

This paper presents fully ab initio calculations of the broadenings and spectral shapes of O-2 infrared absorption lines in a free gas and when confined in nanoporous media. These calculations are performed, without use of any adjusted parameter, using a recently proposed approach [Phys. Rev. A 87, 013403 (2013)] that is based on requantized classical molecular-dynamics simulations. This involves studying the time evolutions of the translational and rotational motions of large numbers of molecules taking molecule-molecule and molecule-surface collisions into account through realistic interaction potentials. These simulations enable predictions of dipole autocorrelation functions whose Fourier-Laplace transforms yield the associated spectra. Comparisons are then made with broadening coefficients and line shapes provided by new and previous experiments. The good agreement between calculated and measured results confirms the veracity of the proposed model for a free gas and shows that the effects of confinement, which induce significant modifications to the line shapes, are correctly predicted. The need for improved characterization of the shape and size of pores in random nanoporous media is highlighted. DOI: 10.1103/PhysRevA.87.032510 (Less)

We show that using upconverting nanoparticles in diffuse luminescence imaging yields superior image resolutions as compared with current fluorophores and highlight the importance of proper optical characterization of them for in-vivo imaging.

We have accomplished deep tissue optical imaging of upconverting nanoparticles at 800 nm, using millisecond single pulse excitation with high peak power. This is achieved by carefully choosing the pulse parameters, derived from time-resolved rate-equation analysis, which result in higher intrinsic quantum yield that is utilized by upconverting nanoparticles for generating this near infrared upconversion emission. The pulsed excitation approach thus promises previously unreachable imaging depths and shorter data acquisition times compared with continuous wave excitation, while simultaneously keeping the possible thermal side-effects of the excitation light moderate. These key results facilitate means to break through the general shallow depth limit of upconverting-nanoparticle-based fluorescence techniques, necessary for a range of biomedical applications, including diffuse optical imaging, photodynamic therapy and remote activation of biomolecules in deep tissues.

PURPOSE. The aims of this study were to use transscleral optical spectroscopy to analyze normal and tumor-infiltrated areas of enucleated human eyes, and to characterize the spectral properties of uveal melanomas in relation to various morphological features. METHODS. Nine consecutive eyes enucleated for uveal melanoma were examined by transscleral spectroscopy, using a fiber-optic probe that exerted a fixed pressure on the scleral surface. Spectroscopic measurements, covering the wavelength range of 400-1100 nm, were sequentially performed over the uveal melanoma and on the opposite (normal) side of each eye. The eyes were then processed for histological and immunohistochemical analyses. Comparisons between spectral and morphological parameters were performed by Spearman's rank correlation coefficient and unpaired t-test. RESULTS. The average reflection intensity obtained from the normal side of the eyes was higher than that from the tumors. The spectral imprint of hemoglobin was lower and that of water was considerably stronger when compared with the tumor side. The diffuse reflection spectra from the melanomas showed a strong correlation with the degree of tumor pigmentation (Spearman's rho = -0.87, P < 0.0001). A weaker correlation was observed between the amount of hemoglobin-related absorption and the density of intratumoral blood vessels (Spearman's rho = -0.25, P = 0.023). The mean diffuse reflection intensity obtained from the spindle cell melanomas was significantly higher than that from the mixed and epithelioid cell melanomas (P < 0.0001). CONCLUSIONS. Although future in vivo studies are required, these data suggest that transscleral optical spectroscopy is a feasible method for identification and morphological assessment of choroidal tumors. (Invest Ophthalmol Vis Sci. 2012;53:5379-5385) DOI: 10.1167/iovs.12-9840 (Less)

Gas cells made of porous materials have been applied in multimode diode laser correlation spectroscopy measurements of oxygen. A pathlength enhancement of more than one order of magnitude compared with an open-air setup was achieved.

Upconverting nanoparticles have recently drawn increasingly attention as contrast agents for optical bioimaging. They enable autofluorescence-free imaging within the tissue optical window, and improved spatial resolution as compared to conventional fluorescence-based contrast agents.

Wall-collision broadening of near-infrared absorption lines of molecular oxygen confined in nanoporous zirconia is studied by employing high-resolution diode-laser spectroscopy. The broadening is studied for pores of different sizes under a range of pressures, providing new insights on how wall collisions and intermolecular collisions influence the total spectroscopic line profile. The pressure series show that wall-collision broadening is relatively more prominent under reduced pressures, enabling sensitive means to probe pore sizes of porous materials. In addition, we show that the total wall-collision-broadened profile strongly deviates from a Voigt profile and that wall-collision broadening exhibits an additive-like behavior to the pressure and Doppler broadening.

Spectroscopic gas sensing is both a commercial success and a rapidly advancing scientific field. Throughout the years, massive efforts have been directed towards improving detection limits by achieving long interaction pathlengths. Prominent examples include the use of conventional multipass gas cells, sophisticated high-finesse cavities, gas-filled holey fibers, integrating spheres, and diffusive reflectors. Despite this rich flora of approaches, there is a continuous struggle to reduce size, gas volume, cost and alignment complexity. Here, we show that extreme light scattering in porous materials can be used to realise miniature gas cells. Near-infrared transmission through a 7 mm zirconia (ZrO2) sample with a 49% porosity and subwavelength pore structure (on the order of 100 nm) gives rise to an effective gas interaction pathlength above 5 meters, an enhancement corresponding to 750 passes through a conventional multipass cell. This essentially different approach to pathlength enhancement opens a new route to compact, alignment-free and low-cost optical sensor systems.

Accurate quantification of photosensitizers is in many cases a critical issue in photodynamic therapy. As a noninvasive and sensitive tool, fluorescence imaging has attracted particular interest for quantification in pre-clinical research. However, due to the absorption of excitation and emission light by turbid media, such as biological tissue, the detected fluorescence signal does not have a simple and unique dependence on the fluorophore concentration for different tissues, but depends in a complex way on other parameters as well. For this reason, little has been done on drug quantification in vivo by the fluorescence imaging technique. In this paper we present a novel approach to compensate for the light absorption in homogeneous turbid media both for the excitation and emission light, utilizing time-resolved fluorescence white Monte Carlo simulations combined with the Beer-Lambert law. This method shows that the corrected fluorescence intensity is almost proportional to the absolute fluorophore concentration. The results on controllable tissue phantoms and murine tissues are presented and show good correlations between the evaluated fluorescence intensities after the light-absorption correction and absolute fluorophore concentrations. These results suggest that the technique potentially provides the means to quantify the fluorophore concentration from fluorescence images.

To study the feasibility of using transscleral visible/near-infrared spectroscopy (Vis/NIRS) to estimate the content of haemoglobin in choroidal tumour phantoms of ex vivo porcine eyes.Thirty enucleated porcine eyes were prepared with a tumour phantom made by injecting a suspension of gelatine, titanium dioxide and human blood into the suprachoroidal space. The blood concentrations used were 2.5%, 25% and 50%, with 10 eyes in each group. Alternating Vis/NIRS measurements were taken over the phantom inclusion and on the opposite (normal) side of each eye. For statistical analysis, a genetic algorithm was utilized to suppress insignificant wavelengths in the spectra. The processed spectra were then used to build a regression model based on partial least squares regression and evaluated by twofold cross-validation.Ultrasonography revealed that all phantoms were localized within the suprachoroidal space with no penetration through the retina. The largest mean diameters of the phantoms with 2.5%, 25% and 50% blood were 15.5, 15.2 and 15.7 mm, respectively (p0.05). The largest mean thicknesses were 4.5, 4.5 and 4.8 mm, respectively (p0.05). Statistical analysis of the spectral data showed that it was possible to correctly discriminate between the normal side and the tumour phantom side of the eyes in 99.88% of cases. The phantoms could be correctly classified according to their blood concentrations in 99.42% of cases.This study demonstrates that transscleral Vis/NIRS is a feasible and accurate method for the detection of choroidal tumours and to assess the haemoglobin content in such lesions.

The main broadening mechanisms of absorption lines in free gas are known to be the pressure-and-Doppler broadening. However, with gas trapped within nanoporous materials, wall-collision broadening may become non-negligible. We present and discuss experimental data which show the effect of wall-collision broadening and its applications.

Fluorescence diffuse optical tomography is known to be a highly ill-posed problem. We demonstrate methods to exploit the nonlinearity of upconverting fluorophores to significantly improve the quality and amount of the input data. Furthermore, we show that it is possible to obtain reconstructions of higher resolutions using nonlinear fluorophores.

Fluorescence diffuse imaging suffers from limited spatial resolution. In this letter, we report a scanning imaging approach to increase the resolution of fluorescence diffuse imaging using nonlinear fluorophores. The resolution of a linear flurophore was compared with nonlinear upconverting nanoparticles (NaYF4 : Yb3+ /Tm3+ ) in a tissue phantom. A resolution improvement of a factor of 1.3 was found experimentally. Simulations suggested a maximum resolution improvement of a factor of 1.45. Usage of nonlinear fluorophores is a promising method for increasing the spatial resolution in fluorescence diffuse imaging. c 2010 Optical Society of America

A method to exploit the nonlinearity of upconverting nanoparticles to increase information quantity in fluorescence diffuse optical tomography, by including excitation with two beams simultaneously, is demonstrated. The increased information resulted in more accurate reconstructions.

Optical spectroscopy has been used as a supplement to conventional techniques for analyzing and diagnosing cancer in many human organs. Because ocular tumors may be characterized by their different melanin content, we investigated the feasibility of using transscleral visible/near-infrared spectroscopy (Vis/NIRS) to estimate the quantity of melanin in a novel uveal melanoma phantom of ex vivo porcine eyes. The phantoms were made by injecting a freshly prepared suspension of 15% (wt/vol) gelatin, 10 mg/ml titanium dioxide (TiO(2)), and natural melanin, isolated from the ink sac of cuttlefish (Sepia officinalis), into the suprachoroidal space of 30 enucleated porcine eyes. The melanin concentrations used were 1 mg/ml, 2 mg/ml, and 3 mg/ml, with 10 eyes in each group. After gelation, the size and location of the phantoms were documented by B-scan ultrasonography and transillumination. Vis/NIRS recordings, covering the wavelength region from 550 to 1000 nm, were performed with two optical fibers separated by 6 mm to deliver and collect the light through the sclera. During all measurements, the exact pressure exerted by the fiber probe on the scleral surface was monitored by placing the eye on an electronic scale. Transscleral Vis/NIRS was performed across the phantom inclusion, as well as on the opposite (normal) side of each eye. A total of three consecutive measurements were carried out alternately on each side of the globe. The spectral data were analyzed using partial least squares regression. In the melanin concentration groups of 1 mg/ml (n = 10), 2 mg/ml (n = 10), and 3 mg/ml (n = 10), the largest basal phantom diameters (mean +/- SD) were 14.9 +/- 1.6 mm, 14.6 +/- 1.5 mm, and 14.3 +/- 1.0 mm, respectively (p > 0.05). The largest phantom thicknesses (mean +/- SD) were 4.0 +/- 0.5 mm, 4.4 +/- 0.7 mm, and 4.5 +/- 0.5 mm, respectively (p > 0.05). Statistical regression modeling of the Vis/NIRS data revealed that it was possible to correctly classify the phantoms according to their melanin concentrations in 84.4% of cases. The correct classification rate for phantoms with the lowest (1 mg/ml) and highest (3 mg/ml) melanin concentrations was 99.2%. The study demonstrates that transscleral Vis/NIRS is a feasible and accurate method for predicting the content of melanin in choroidal lesions.

Fluorescence diffuse optical tomography (FDOT) can provide important information in biomedical studies. In this ill-posed problem, suppression of background tissue autofluorescence is of utmost importance. We report a method for autofluorescence-insensitive FDOT using nonlinear upconverting nanoparticles (NaYF4:Yb3+/Tm3+) in a tissue phantom under excitation intensities well below tissue-damage thresholds. Even with the intrinsic autofluorescence from the phantom only, the reconstruction of the nanoparticles is of much better quality than the reconstruction of a Stokes-shifting dye. In addition, the nonlinear power dependence leads to more confined reconstructions and may increase the resolution in FDOT.

We report on improved image detectability for fluorescence diffuse optical tomography using upconverting nanoparticles doped with rare-earth elements. Core-shell NaYF4 : Yb3+/Er3+ @ NaYF4 upconvert ...

The purpose of this study was to investigate how the geometry of a fiber optic probe affects the transmission and reflection of light through the scleral eye wall. Two geometrical parameters of the fiber probe were investigated: the source-detector distance and the fiber protrusion, i.e. the length of the fiber extending from the flat surface of the fiber probe. For optimization of the fiber optic probe geometry, fluorescence stained choroidal tumor phantoms in ex vivo porcine eyes were measured with both diffuse reflectance- and laser-induced fluorescence spectroscopy. The strength of the fluorescence signal compared to the excitation signal was used as a measure for optimization. Intraocular pressure (IOP) and temperature were monitored to assess the impact of the probe on the eye. For visualizing any possible damage caused by the probe, the scleral surface was imaged with scanning electron microscopy after completion of the spectroscopic measurements. A source-detector distance of 5 mm with zero fiber protrusion was considered optimal in terms of spectroscopic contrast, however, a slight fiber protrusion of 0.5 mm is argued to be advantageous for clinical measurements. The study further indicates that transscleral spectroscopy can be safely performed in human eyes under in vivo conditions, without leading to an unacceptable IOP elevation, a significant rise in tissue temperature, or any visible damage to the scleral surface.

Fluorescence diffuse optical tomography (FDOT) is a biomedical imaging modality that can be used for localization and quantification of fluorescent molecules inside turbid media. In this ill-posed problem, the reconstruction quality is directly determined by the amount and quality of the information obtained from the boundary measurements. Regularly, more information can be obtained by increasing the number of excitation positions in an FDOT system. However, the maximum number of excitation positions is limited by the finite size of the excitation beam. In the present work, we demonstrate a method in FDOT to exploit the unique nonlinear power dependence of upconverting nanoparticles to further increase the amount of information in a raster-scanning setup by including excitation with two beams simultaneously. We show that the additional information can be used to obtain more accurate reconstructions.

Autofluorescence is a nuisance in the field of fluorescence imaging and tomography of exogenous molecular markers in tissue, degrading the quality of the collected data. In this letter, we report autofluorescence insensitive imaging using highly efficient upconverting nanocrystals (NaYF4: Yb3+ /Tm3+) in a tissue phantom illuminated with near- infrared radiation of 85 mW/cm(2). It was found that imaging with such nanocrystals leads to an exceptionally high contrast compared to traditional downconverting fluorophores due to the absence of autofluorescence. Upconverting nanocrystals may be envisaged as important biological markers for tissue imaging purposes. c 2008 American Institute of Physics. [DOI: 10.1063/1.3005588]

Deep tissue excitation of upconverting nanoparticles is limited for biomedical applications by water absorption. By modifying the nanoparticles to shift the excitation wavelength, we demonstrate better depth sensitivity.