Peter D. Kaplan

Two-photon 3-D mapping of ex vivo human skin endogenous fluorescence species based on fluorescence emission spectra

Spectral resolved tissue imaging has a broad range of biomedical applications such as the minimally invasive diagnosis of diseases and the study of wound healing and tissue engineering processes. Two-photon microscopy imaging of endogenous fluorescence has been shown to be a powerful method for the quantification of tissue structure and biochemistry. While two-photon excited autofluorescence is observed ubiquitously, the identities and distributions of endogenous fluorophores have not been completely characterized in most tissues. We develop an image-guided spectral analysis method to analyze the distribution of fluorophores in human skin from 3-D resolved two-photon images. We identify five factors that contribute to most of the luminescence signals from human skin. Luminescence species identified include tryptophan, NAD(P)H, melanin, and elastin, which are autofluorescent, and collagen that contributes to a second harmonic signal.

Entropically driven self–assembly and interaction in suspension

In this paper we present fundamental studies elucidating the role of entropy in particle suspensions. We focus on systems composed of large colloidal particles along with a second, usually smaller species such as a particle or polymer. We describe direct measurements of these interactions in suspension, and we systematically show how these forces can be used to control the self–assembly of colloidal particles. The paper provides a unified review of the experiments from our laboratory, and in a few cases touches on very recent results.

Geometric constraints for the design of diffusing-wave spectroscopy experiments.

Diffusing-wave spectroscopy (DWS) experiments require the choice of suitable sample geometry. We study sample geometries for transmission experiments by performing DWS measurements on a variable thickness cell. The data reveal that DWS works well, giving consistent answers to within 5% when the cell is more than 10 random walk step lengths thick, and that the input geometry is less significant when sample cells are immersed in water than when they are surrounded by air. Further, we see that the applicability of the diffusion approximation depends on the anisotropy of individual scattering events.

Optical coherence tomography of skin for measurement of epidermal thickness by shapelet-based image analysis

Optical coherence tomography (OCT) provides a non-invasive method for in-vivo imaging of sub-surface skin tissue. Many skin features such as sweat glands and blisters are clearly observable in OCT images. It seems therefore probable that OCT could be used for the detection and identification of lesions and skin cancers. These applications, however, have not been well developed. One area in dermatology where OCT has been applied is the measurement of epidermal thickness. OCT images are inherently noisy and measurements based on them require intensive manual processing. A robust method to automatically detect and measure features of interest is necessary to enable routine application of OCT. As a first step, we approach the seemingly straightforward problem of measuring epidermal thickness. In this paper we describe a novel shapelet-based image processing technique for the automatic identification of the upper and lower boundaries of the epidermis in living human skin tissue. These boundaries are used to measure epidermal thickness. To our knowledge, this is the first report of automated feature identification and measurement from OCT images of skin.

Microscope-based static light-scattering instrument

We describe a new design for a microscope-based static light-scattering instrument that provides simultaneous high-resolution images and static light-scattering data. By correlating real space images with scattering patterns, we can interpret measurements from heterogeneous samples, which we illustrate by using biological tissue.

Microscopic origin of light scattering in tissue.

A newly designed instrument, the static light-scattering (SLS) microscope, which combines light microscopy with SLS, enables us to characterize local light-scattering patterns of thin tissue sections. Each measurement is performed with an illumination beam of 70-microm diameter. On these length scales, tissue is not homogeneous. Both structural ordering and small heterogeneities contribute to the scattering signal. Raw SLS data consist of a two-dimensional intensity distribution map I(theta, phi), showing the dependence of the scattered intensity I on the scattering angle theta and the azimuthal angle phi. In contrast to the majority of experiments and to simulations that consider only the scattering angle, we additionally perform an analysis of the azimuthal dependence I(phi). We estimate different contributions to the azimuthal scattering variation and show that a significant fraction of the azimuthal amplitude is the result of tissue structure. As a demonstration of the importance of the structure-dependent part of the azimuthal signal, we show that this function of the scattered light alone can be used to classify tissue types with surprisingly high specificity and sensitivity.

Light-scattering microscope

We demonstrate a new design for a light-scattering microscope that is convenient to use and that allows simultaneous imaging and light scattering. The design is motivated by the growing use of thermal fluctuations to probe the viscoelastic properties of complex inhomogeneous environments. We demonstrate measurements of an optically nonergodic sample, one of the most challenging light-scattering applications.

Two-photon 3D mapping of tissue endogenous fluorescence species based on fluoresence excitation spectra

Deep tissue imaging may have important biomedical applications in the areas of skin disease diagnosis, wound healing, and tissue engineering. For the study oftissue physiology with microscopic resolution, we used two-photon microscopic imaging based on the excitation of endogenous fluorophores. While autofluorescence is observed ubiquitously in many tissue types, the identities and distributions of these fluorophores have not been completely characterized. The different fluorescent species are expected to have different fluorescence excitation and emission spectra. Self-modeling curve resolution (SMCR) can be applied to extract spectroscopic components from two-photon images. In ex vivo human skin, we were able to acquire a four-dimensional data set (3D space + excitation spectra). We extracted the major spectral components from this data set using multivariate curve resolution and correlated these species with known tissue structures. From the SMCR analysis, it was determined that there are approximately seven factors that contribute to most of the autofluorescence from human skin. This analysis provides us with the concentration ofthe species at different depths within the skin and also with a reconstructed image of the skin due to each single factor alone. Several ofthese chemical components have been identified, such as collagen, elastin, and NAD(P)H. In addition to providing insight into tissue physiology, we are able to optimize the excitation wavelength for each biochemical species for skin imaging applications.

Two-photon 3D mapping of tisssue endogenous fluorescence species based on fluorescence emission spectra

Two-photon microscopy imaging of endogenous fluorescence has been shown to be a powerful method for the quantification of tissue structure and biochemistry. While autofluorescence is observed in many tissue types, the identities and distributions of these fluorophores have not been completely characterized. Image guided spectral analysis is being developed to aid in extracting spectroscopic components from two-photon images. This methodology is being applied to the study of human skin. In ex vivo specimens, the overall bulk emission spectrum of the skin, the layer-resolved emission spectra of the stratum corneum, stratum spinosum, basal layer, and dermis, and the emission spectra of surgically exposed dermis have been measured. From the image guided spectral analysis, it was determined that there are approximately five factors that contribute to most of the luminescence signals from human skin. The autofluorescent species identified include tryptophan, NAD(P)H, melanin (or localizing species), and elastin. The collagen matrix contributes to a second harmonic signal.

Applications of two-photon fluorescence microscopy in deep-tissue imaging

Based on the non-linear excitation of fluorescence molecules, two-photon fluorescence microscopy has become a significant new tool for biological imaging. The point-like excitation characteristic of this technique enhances image quality by the virtual elimination of off-focal fluorescence. Furthermore, sample photodamage is greatly reduced because fluorescence excitation is limited to the focal region. For deep tissue imaging, two-photon microscopy has the additional benefit in the greatly improved imaging depth penetration. Since the near- infrared laser sources used in two-photon microscopy scatter less than their UV/glue-green counterparts, in-depth imaging of highly scattering specimen can be greatly improved. In this work, we will present data characterizing both the imaging characteristics (point-spread-functions) and tissue samples (skin) images using this novel technology. In particular, we will demonstrate how blind deconvolution can be used further improve two-photon image quality and how this technique can be used to study mechanisms of chemically-enhanced, transdermal drug delivery.