Diffusive and directional intracellular dynamics measured by field-based dynamic light scattering.

Quantitative measurement of diffusive and directional processes of intracellular structures is not only critical in understanding cell mechanics and functions, but also has many applications, such as investigation of cellular responses to therapeutic agents. We introduce a label-free optical technique that allows non-perturbative characterization of localized intracellular dynamics. The method combines a field-based dynamic light scattering analysis with a confocal interferometric microscope to provide a statistical measure of the diffusive and directional motion of scattering structures inside a microscopic probe volume. To demonstrate the potential of this technique, we examined the localized intracellular dynamics in human epithelial ovarian cancer cells. We observed the distinctive temporal regimes of intracellular dynamics, which transitions from random to directional processes on a timescale of ~0.01 sec. In addition, we observed disrupted directional processes on the timescale of 1 approximately 5 sec by the application of a microtubule polymerization inhibitor, Colchicine, and ATP depletion.

T helper type 1 cytokines and keratinocyte growth factor play a critical role in pseudoepitheliomatous hyperplasia initiation during cutaneous leishmaniasis.

Pseudoepitheliomatous hyperplasia (PEH) is an exuberant proliferation of the epidermis. The underlying mechanism(s) that lead to PEH have not been completely elucidated. Here, we characterize PEH during the healing stages of cutaneous leishmanial ulcers in mice. During experimental cutaneous leishmaniasis (CL) C57BL/6 mice produce PEH, and BALB/c do not. A series of immunohistochemical and immunological studies were performed to identify the secretory products of PEH regulation. We observed that the distribution of TNF-alpha and IFN-gamma under PEH had a stripe-like diffuse pattern and localized in the upper part of the papillary dermis directly under the proliferating epidermis. Macrophages were identified as the major source of TNF-alpha (56.3%). The importance of IFN-gamma and TNF-alpha in PEH development was proven by the initiation of PEH after three intralesional injections of TNF-alpha and IFN-gamma every three days in infected BALB/c mice. In C57BL/6 mice, keratinocyte growth factor (KGF) expressing cells were found immediately under the basal membrane of the hyperplastic epidermis in comparison with sporadic KGF positive cells deep in the dermis of BALB/c mice. Quantitative RT-PCR analysis demonstrated increased KGF and KGF receptor expression in uninfected C57BL/6 mice as compared to BALB/c mice. These data indicate that Th1 cytokines and KGF play a critical role in PEH initiation during CL.

Light-induced retinal vascular damage by Pd-porphyrin luminescent oxygen probes.

PURPOSE: The phosphorescence lifetime of certain metalloporphyrins dissolved in a physiological medium provides an optical signature for local oxygen concentration (pO(2)). This effect is used for measuring physiological pO(2) levels in various tissues. However, the phosphorescence quenching of certain metalloporphyrin triplet states by oxygen also creates singlet oxygen, which is highly reactive and capable of inducing tissue damage. In the current study, the Pd-meso-tetra(4-carboxyphenyl) porphyrin dye (PdTCPP) was simultaneously used as an oxygen sensor and a photosensitizer. Phototoxicity was assessed in the eye fundus and correlated with tissue oxygenation, drug-light dose, and severity of tissue damage. METHODS: The kinetics of photochemical oxygen depletion during PdTCPP excitation was measured in vivo on the optic disc of piglets by phosphorescence lifetime imaging. Blood-retinal barrier breakdown and tissue damage were assessed by confocal and electron microscopy. RESULTS: For a retinal irradiance of 5 mW/cm(2) at 532 nm and an injected PdTCPP dose of 20 mg/kg, the mean phosphorescence lifetime measured at the optic disc increased from 100 to 600 micros within 8 minutes of continuous illumination. This corresponds to a decrease of pO(2) from 25 to 0 mm Hg, induced by a light dose of only 2.4 J/cm(2). An exposure time of 6 minutes (1.8 J/cm(2)) generated an increase in phosphorescence lifetime from 100 to 400 micros, corresponding to a decrease in pO(2) from 25 to 4 mm Hg. This caused edema in all retinal layers, whereas irradiation of 2 minutes (0.6 J/cm(2)) damaged blood vessels and induced edema in the inner nuclear layer only. Heavy redistribution of occludin occurred after a 30-minute exposure time (9 J/cm(2)). CONCLUSIONS: PdTCPP is potentially phototoxic under certain experimental conditions and can induce damage in peripapillary retina and optic nerve head after light exposure. The severity of tissue damage correlates with the phosphorescence measurements.