Liquid phantoms for near-infrared and diffuse correlation spectroscopies with tunable optical and dynamic properties.

We present the recipe and characterization for preparing liquid phantoms that are suitable for both near-infrared spectroscopy and diffuse correlation spectroscopy. The phantoms have well-defined and tunable optical and dynamic properties, and consist of a solution of water and glycerol with fat emulsion as the scattering element. The recipe takes into account the effect of bulk refractive index changes due to the addition of glycerol, which is commonly used to alter the sample viscosity.

Stability of gel wax based optical scattering phantoms.

Phantoms with tuneable optical scattering properties are essential in the development and refinement of optical based imaging techniques. Mineral oil based 'gel wax' phantoms are the subject of increasing interest due to their ease and speed of manufacture, non-toxic nature, ability to cast into anatomically realistic shapes, as well as their cost-effective nature of production. The addition of scatterers such as titanium dioxide powder and monodisperse silica microspheres to the gel wax allows for the creation of phantoms with a controllable optical scattering coefficient. To enable repeated use of such phantoms, the stability of the scattering properties must be determined-a property which has yet to be investigated. We present an analysis of the stability of the reduced scattering coefficient ( µ s ' ) of such phantoms over time. We conclude that due to the measurable reduction in scattering coefficient over time, gel wax phantoms embedded with silica spheres may not be suitable for repeated use over time, however gel wax-TiO2 phantoms are much more temporally stable.

Characterizing the optical properties of human brain tissue with high numerical aperture optical coherence tomography.

Quantification of tissue optical properties with optical coherence tomography (OCT) has proven to be useful in evaluating structural characteristics and pathological changes. Previous studies primarily used an exponential model to analyze low numerical aperture (NA) OCT measurements and obtain the total attenuation coefficient for biological tissue. In this study, we develop a systematic method that includes the confocal parameter for modeling the depth profiles of high NA OCT, when the confocal parameter cannot be ignored. This approach enables us to quantify tissue optical properties with higher lateral resolution. The model parameter predictions for the scattering coefficients were tested with calibrated microsphere phantoms. The application of the model to human brain tissue demonstrates that the scattering and back-scattering coefficients each provide unique information, allowing us to differentially identify laminar structures in primary visual cortex and distinguish various nuclei in the midbrain. The combination of the two optical properties greatly enhances the power of OCT to distinguish intricate structures in the human brain beyond what is achievable with measured OCT intensity information alone, and therefore has the potential to enable objective evaluation of normal brain structure as well as pathological conditions in brain diseases. These results represent a promising step for enabling the quantification of tissue optical properties from high NA OCT.

Augmented line-scan focal modulation microscopy for multi-dimensional imaging of zebrafish heart in vivo.

Multi-dimensional fluorescence imaging of live animal models demands strong optical sectioning, high spatial resolution, fast image acquisition, and minimal photobleaching. While conventional laser scanning microscopes are capable of deep penetration and sub-cellular resolution, they are generally too slow and causing excessive photobleaching for volumetric or time-lapse imaging. We demonstrate the performance of an augmented line-scan focal modulation microscope (aLSFMM), a high-speed imaging platform that affords above video-rate imaging speed by the use of line scanning. Exceptional background rejection is accomplished by combining a confocal slit with focal modulation. The image quality is further improved by merging the information from simultaneously acquired focal modulation and confocal images. Such a hybrid imaging scheme makes it possible to use very low power excitation light in high-speed imaging, and therefore leads to reduced photobleaching that is desirable for three-dimensional (3D) and four-dimensional (4D) in vivo image acquisition.

Development of thin skin mimicking bilayer solid tissue phantoms for optical spectroscopic studies.

In vivo spectroscopic measurements have the proven potential to provide important insight about the changes in tissue during the development of malignancies and thus help to diagnose tissue pathologies. Extraction of intrinsic data in the presence of varying amounts of scatterers and absorbers offers great challenges in the development of such techniques to the clinical level. Fabrication of optical phantoms, tailored to the biochemical as well as morphological features of the target tissue, can help to generate a spectral database for a given optical spectral measurement system. Such databases, along with appropriate pattern matching algorithms, could be integrated with in vivo measurements for any desired quantitative analysis of the target tissue. This paper addresses the fabrication of such soft, photo stable, thin bilayer phantoms, mimicking skin tissue in layer dimensions and optical properties. The performance evaluation of the fabricated set of phantoms is carried out using a portable fluorescence spectral measurement system. The alterations in flavin adenine dinucleotide (FAD)-a tissue fluorophore that provides important information about dysplastic progressions in tissues associated with cancer development based on changes in emission spectra-fluorescence with varied concentrations of absorbers and scatterers present in the phantom are analyzed and the results are presented. Alterations in the emission intensity, shift in emission wavelength and broadening of the emission spectrum were found to be potential markers in the assessment of biochemical changes that occur during the progression of dysplasia.

Evaluating platelet aggregation dynamics from laser speckle fluctuations.

Platelets are key to maintaining hemostasis and impaired platelet aggregation could lead to hemorrhage or thrombosis. We report a new approach that exploits laser speckle intensity fluctuations, emanated from a drop of platelet-rich-plasma (PRP), to profile aggregation. Speckle fluctuation rate is quantified by the speckle intensity autocorrelation, g2(t), from which the aggregate size is deduced. We first apply this approach to evaluate polystyrene bead aggregation, triggered by salt. Next, we assess dose-dependent platelet aggregation and inhibition in human PRP spiked with adenosine diphosphate and clopidogrel. Additional spatio-temporal speckle analyses yield 2-dimensional maps of particle displacements to visualize platelet aggregate foci within minutes and quantify aggregation dynamics. These findings demonstrate the unique opportunity for assessing platelet health within minutes for diagnosing bleeding disorders and monitoring anti-platelet therapies.

Experimental results of full scattering profile from finger tissue-like phantom.

Human tissue is one of the most complex optical media since it is turbid and nonhomogeneous. We suggest a new optical method for sensing physiological tissue state, based on the collection of the ejected light at all exit angles, to receive the full scattering profile. We built a unique set-up for noninvasive encircled measurement. We use a laser, a photodetector and finger tissues-mimicking phantoms presenting different optical properties. Our method reveals an isobaric point, which is independent of the optical properties. We compared the new finger tissues-like phantoms to others samples and found the linear dependence between the isobaric point's angle and the exact tissue geometry. These findings can be useful for biomedical applications such as non-invasive and simple diagnostic of the fingertip joint, ear lobe and pinched tissues.

Analyzing spatial correlations in tissue using angle-resolved low coherence interferometry measurements guided by co-located optical coherence tomography.

Angle-resolved low coherence interferometry (a/LCI) is an optical technique used to measure nuclear morphology in situ. However, a/LCI is not an imaging modality and can produce ambiguous results when the measurements are not properly oriented to the tissue architecture. Here we present a 2D a/LCI system which incorporates optical coherence tomography imaging to guide the measurements. System design and characterization are presented, along with example cases which demonstrate the utility of the combined measurements. In addition, future development and applications of this dual modality approach are discussed.

Depth-resolved measurements with elliptically polarized reflectance spectroscopy.

The ability of elliptical polarized reflectance spectroscopy (EPRS) to detect spectroscopic alterations in tissue mimicking phantoms and in biological tissue in situ is demonstrated. It is shown that there is a linear relationship between light penetration depth and ellipticity. This dependence is used to demonstrate the feasibility of a depth-resolved spectroscopic imaging using EPRS. The advantages and drawbacks of EPRS in evaluation of biological tissue are analyzed and discussed.

In vivo assessment of optical properties of basal cell carcinoma and differentiation of BCC subtypes by high-definition optical coherence tomography.

High-definition optical coherence tomography (HD-OCT) features of basal cell carcinoma (BCC) have recently been defined. We assessed in vivo optical properties (IV-OP) of BCC, by HD-OCT. Moreover their critical values for BCC subtype differentiation were determined. The technique of semi-log plot whereby an exponential function becomes linear has been implemented on HD-OCT signals. The relative attenuation factor (µraf ) at different skin layers could be assessed.. IV-OP of superficial BCC with high diagnostic accuracy (DA) and high negative predictive values (NPV) were (i) decreased µraf in lower part of epidermis and (ii) increased epidermal thickness (E-T). IV-OP of nodular BCC with good to high DA and NPV were (i) less negative µraf in papillary dermis compared to normal adjacent skin and (ii) significantly decreased E-T and papillary dermal thickness (PD-T). In infiltrative BCC (i) high µraf in reticular dermis compared to normal adjacent skin and (ii) presence of peaks and falls in reticular dermis had good DA and high NPV. HD-OCT seems to enable the combination of in vivo morphological analysis of cellular and 3-D micro-architectural structures with IV-OP analysis of BCC. This permits BCC sub-differentiation with higher accuracy than in vivo HD-OCT analysis of morphology alone.

Application of full field optical studies for pulsatile flow in a carotid artery phantom.

A preliminary comparative measurement between particle imaging velocimetry (PIV) and laser speckle contrast analysis (LASCA) to study pulsatile flow using ventricular assist device in a patient-specific carotid artery phantom is reported. These full-field optical techniques have both been used to study flow and extract complementary parameters. We use the high spatial resolution of PIV to generate a full velocity map of the flow field and the high temporal resolution of LASCA to extract the detailed frequency spectrum of the fluid pulses. Using this combination of techniques a complete study of complex pulsatile flow in an intricate flow network can be studied.

Experimental system for measuring the full scattering profile of circular phantoms.

Optical methods for monitoring physiological tissue state are important and useful because they are non-invasive and sensitive. Experimental measurements of the full scattering profile of circular phantoms are presented. We report, for the first time, an experimental observation of a typical reflected light intensity behavior for a circular structure characterized by the isobaric point. We previously suggested a new theoretically method for measuring the full scattering profile, which is the angular distribution of light intensity, of cylindrical tissues. In this work we present that the experimental result match the simulation results. We show the isobaric point at 105° for a cylindrical phantom with a 7mm diameter, while for a 16mm diameter phantom the isobaric point is at 125°. Furthermore, the experimental work present a new crossover point of the full scattering profiles of subjects with different diameters of the cylindrical tissues.

Accessing to arteriovenous blood flow dynamics response using combined laser speckle contrast imaging and skin optical clearing.

Laser speckle contrast imaging (LSCI) shows a great potential for monitoring blood flow, but the spatial resolution suffers from the scattering of tissue. Here, we demonstrate the capability of a combination method of LSCI and skin optical clearing to describe in detail the dynamic response of cutaneous vasculature to vasoactive noradrenaline injection. Moreover, the superior resolution, contrast and sensitivity make it possible to rebuild arteries-veins separation and quantitatively assess the blood flow dynamical changes in terms of flow velocity and vascular diameter at single artery or vein level.

Numerical investigation of two-dimensional light scattering patterns of cervical cell nuclei to map dysplastic changes at different epithelial depths.

We use an extensive set of quantitative histopathology data to construct realistic three-dimensional models of normal and dysplastic cervical cell nuclei at different epithelial depths. We then employ the finite-difference time-domain method to numerically simulate the light scattering response of these representative models as a function of the polar and azimuthal scattering angles. The results indicate that intensity and shape metrics computed from two-dimensional scattering patterns can be used to distinguish between different diagnostic categories. Our numerical study also suggests that different epithelial layers and angular ranges need to be considered separately to fully exploit the diagnostic potential of two-dimensional light scattering measurements.

Sub-diffusive scattering parameter maps recovered using wide-field high-frequency structured light imaging.

This study investigates the hypothesis that structured light reflectance imaging with high spatial frequency patterns [Formula: see text] can be used to quantitatively map the anisotropic scattering phase function distribution [Formula: see text] in turbid media. Monte Carlo simulations were used in part to establish a semi-empirical model of demodulated reflectance ([Formula: see text]) in terms of dimensionless scattering [Formula: see text] and [Formula: see text], a metric of the first two moments of the [Formula: see text] distribution. Experiments completed in tissue-simulating phantoms showed that simultaneous analysis of [Formula: see text] spectra sampled at multiple [Formula: see text] in the frequency range [0.05-0.5] [Formula: see text] allowed accurate estimation of both [Formula: see text] in the relevant tissue range [0.4-1.8] [Formula: see text], and [Formula: see text] in the range [1.4-1.75]. Pilot measurements of a healthy volunteer exhibited [Formula: see text]-based contrast between scar tissue and surrounding normal skin, which was not as apparent in wide field diffuse imaging. These results represent the first wide-field maps to quantify sub-diffuse scattering parameters, which are sensitive to sub-microscopic tissue structures and composition, and therefore, offer potential for fast diagnostic imaging of ultrastructure on a size scale that is relevant to surgical applications.

Dynamic light scattering from pulsatile flow in the presence of induced motion artifacts.

Continuous health monitoring has become a major theme of our aging society. Portable devices play an important role here. Many optical portable devices are susceptible to motion induced artifacts. We have performed an experimental study for detection of fluid pulsation based on multi-exposure speckle images, in presence of motion induced artifacts. Induced motion of a wide range of frequencies and amplitudes were generated to resemble sensor motion with respect to skin. The data was analyzed using speckle contrast and correlation. We concluded that both techniques have their own advantages, depending on the measurement configuration. A study of angles between illumination and detection revealed that larger angles yields better signal. Shorter exposure time was more successful in extracting the signal. We also performed in-vivo measurements that agree with the in-vitro case. We also show that a minimum collection of two pixels from the speckle image is sufficient to extract relevant results.

A scattering phantom for observing long range order with two-dimensional angle-resolved Low-Coherence Interferometry.

Angle-resolved low coherence interferometry (a/LCI) is an approach for assessing tissue structure based on light scattering data. Recent advances in a/LCI have extended the analysis to study scattering distributions in two dimensions. In order to provide suitable scattering phantoms for 2D a/LCI, we have developed phantoms based on soft lithography which can provide a range of structures including long range order. Here we characterize these phantoms and demonstrate their utility for providing standardized multi-scale structural information for light scattering measurements.

Two-photon excitation in scattering media by spatiotemporally shaped beams and their application in optogenetic stimulation.

The use of wavefront shaping to generate extended optical excitation patterns which are confined to a predetermined volume has become commonplace on various microscopy applications. For multiphoton excitation, three-dimensional confinement can be achieved by combining the technique of temporal focusing of ultra-short pulses with different approaches for lateral light shaping, including computer generated holography or generalized phase contrast. Here we present a theoretical and experimental study on the effect of scattering on the propagation of holographic beams with and without temporal focusing. Results from fixed and acute cortical slices show that temporally focused spatial patterns are extremely robust against the effects of scattering and this permits their three-dimensionally confined excitation for depths more than 500 µm. Finally we prove the efficiency of using temporally focused holographic beams in two-photon stimulation of neurons expressing the red-shifted optogenetic channel C1V1.

Axial response of high-resolution microendoscopy in scattering media.

High-resolution microendoscopy (HRME) uses epi-fluorescence imaging with a coherent fiber-optic bundle to enable in vivo examination of cellular morphology. While the HRME platform has recently gained popularity as a simple alternative to confocal endomicroscopy, the axial response of HRME in thick, scattering tissue has yet to be described quantitatively. These details are important because when analyzing images collected by HRME, out-of-focus light may affect the accuracy of quantitative parameters such as nuclear-to-cytoplasm ratio, which has been proposed as a diagnostic indicator of dysplasia or cancer. In this study we investigated the imaging properties of the HRME system by using phantoms simulating scattering tissue with fluorescently labeled nuclei. We directly compared HRME imaging with confocal endomicroscopy in phantoms and in vivo human tissue. HRME images defocused (deep) objects with apparent diameters and intensity levels that are in agreement with a simple geometric model. Out-of-focus nuclei contribute a relatively low, uniform background level to images which neither leads to the erroneous appearance of large nuclei from deep layers, nor prevents accurate imaging of superficial nuclei with high contrast.

Characterizing optical properties of nano contrast agents by using cross-referencing OCT imaging.

We report a cross-referencing method to quickly and accurately characterize the optical properties of nanoparticles including the extinction, scattering, absorption and backscattering cross sections by using an OCT system alone. Among other applications, such a method is particularly useful for developing nanoparticle-based OCT imaging contrast agents. The method involves comparing two depth-dependent OCT intensity signals collected from two samples (with one having and the other not having the nanoparticles), to extract the extinction and backscattering coefficient, from which the absorption coefficient can be further deduced (with the help of the established scattering theories for predicting the ratio of the backscattering to total scattering cross section). The method has been experimentally validated using test nanoparticles and was then applied to characterizing gold nanocages. With the aid of this method, we were able to successfully synthesize scattering dominant gold nanocages for the first time and demonstrated the highest contrast enhancement ever achieved by the gold nanocages (and by any nanoparticles of a similar size and concentration) in an in vivo mouse tumor model. This method also enables quantitative analysis of contrast enhancement and provides a general guideline on choosing the optimal concentration and optical properties for the nanoparticle-based OCT contrast agents.