Cigarette smoking is the most preventable cause of death in the United States. Researchers have extensively studied
smoking in regards to its association with cancer, cardiovascular, and pulmonary disease. In contrast, the impact of
cigarette smoking on skin has received much less attention. To provide a better understanding of the effect of cigarette
smoking on the human dermal layer, this study used multi-photon microscopy (MPM) to examine collagen in
organotypic skin models exposed to cigarette smoke condensate (CSC). Adult and neonatal organotypic tissue-engineered
artificial skin models (RAFTs) were constructed and exposed to varying concentrations of CSC. Imaging of
the RAFTs was performed using MPM and second-harmonic generation signals (SHG), which allowed for collagen
structure to be viewed and analyzed as well as for collagen density to be assessed from derived depth-dependent decay
(DDD) values. RAFT contraction as related to exposure concentration was monitored as well. Results indicated a dose
dependent between contraction rates and CSC concentration. Collagen structure showed more preservation of its
original structure at a greater depth in RAFTs with higher concentrations of CSC. No clear trends could be drawn from
analysis of derived DDD values.
Tissue patterns appear to be specified by gradients of morphogens. Although it has been established that morphogens are indeed distributed in gradients (morphogenetic gradients), how these gradients arise is not well understood. Two main types of mechanisms have been proposed: (1) diffusion-based, and (2) transcytosis; a third "bucket-brigade" mechanism has also been proposed. The diffusion model is based on the assumption that a morphogen diffuses from a region of origin to form the gradient. The other model proposes that morphogens instead are taken up by one cell and transit that cell to be released on the other side. A third model proposes a “bucket brigade” mechanism in which receptor-bound morphogens on one cell move by being handed off to receptors on an adjacent cell. To provide insight into the mechanism of the formation of the morphogen gradients, we conducted fluorescence correlation spectroscopy (FCS) measurements of morphogen Dpp-EGFP fusion protein in intact Drosophila imaginal disks with two-photon excitation at 850 nm. The FCS results are analyzed with a two-species model. The first species can be attributed to Dpp-EGFP in Brownian motion, while the second species could result from either a large complex involving Dpp-EGFP formed through biochemical reactions, or from anomalous diffusion of Dpp, presumably due to its transport along the cell membranes. Our studies demonstrate for the first time that FCS is capable of investigating molecular dynamics in intact tissues.