Jorge H. Torres

Membrane Tether Formation from Outer Hair Cells with Optical Tweezers

Optical tweezers were used to characterize the mechanical properties of the outer hair cell (OHC) plasma membrane by pulling tethers with 4.5-microm polystyrene beads. Tether formation force and tether force were measured in static and dynamic conditions. A greater force was required for tether formations from OHC lateral wall (499 +/- 152 pN) than from OHC basal end (142 +/- 49 pN). The difference in the force required to pull tethers is consistent with an extensive cytoskeletal framework associated with the lateral wall known as the cortical lattice. The apparent plasma membrane stiffness, estimated under the static conditions by measuring tether force at different tether length, was 3.71 pN/microm for OHC lateral wall and 4.57 pN/microm for OHC basal end. The effective membrane viscosity was measured by pulling tethers at different rates while continuously recording the tether force, and estimated in the range of 2.39 to 5.25 pN x s/microm. The viscous force most likely results from the viscous interactions between plasma membrane lipids and the OHC cortical lattice and/or integral membrane proteins. The information these studies provide on the mechanical properties of the OHC lateral wall is important for understanding the mechanism of OHC electromotility.

Estimation of internal skin temperatures in response to cryogen spray cooling: implications for laser therapy of port wine stains

In many port wine stain (PWS) patients, successful clearing is not achieved even after multiple laser treatments because of inadequate heat generation within the targeted blood vessels. Use of higher radiant exposures has been suggested to improve lesion clearing, but risk of epidermal injury due to nonspecific absorption by melanin increases. It has been demonstrated that cryogen spray cooling (CSC) can protect the epidermis from nonspecific thermal injury during laser treatment of PWS. Inasmuch as epidermal melanin concentration and blood vessel depth vary among patients, evaluation of internal skin temperatures in response to CSC is essential for further development and optimization of treatment parameters on an individual patient basis. We present internal temperature measurements in an epoxy resin phantom in response to CSC and use the results in conjunction with a mathematical model to predict the temperature distribution within human skin for various cooling parameters. Measurements on the epoxy resin phantom show that cryogen film temperature is well below the cryogen boiling point, but a poor thermal contact exists at the cryogen-phantom interface. Based on phantom measurements and model predictions, internal skin temperature reduction remains confined to the upper 400 /spl mu/m for spurt durations as long as 200 ms. At the end of a 100 ms spurt, our results show a 31/spl deg/C temperature reduction at the surface, 12/spl deg/C at a depth of 100 /spl mu/m, and 4/spl deg/C at a depth of 200 /spl mu/m in human skin. Analysis of estimated temperature distributions in response to CSC and temperature profiles obtained by pulsed photothermal radiometry indicates that a significant protective effect is achieved at the surface of laser irradiated PWS skin. Protection of the epidermal basal layer, however, poses a greater challenge when high radiant exposures are used.

Temporal effects of cell adhesion on mechanical characteristics of the single chondrocyte

Cell adhesion to material surfaces is a fundamental phenomenon in tissue response to implanted devices, and an important consideration in tissue engineering. For example, elucidation of phenomena associated with adhesion of chondrocytes to biomaterials is critical in addressing the difficult problem of articular cartilage regeneration. The first objective of this study was to measure the mechanical adhesiveness characteristics of individual rabbit articular chondrocytes as a function of seeding time to provide further understanding of the cell adhesion process. The second objective was to quantify the force required to separate the plasma membrane from the underlying cytoskeleton as a function of seeding time. After culturing chondrocytes on glass coverslips for 1, 2, 4, 6 h, two biomechanical tests were performed on single chondrocytes: (i) mechanical adhesiveness measurement by the cytodetacher; and (ii) plasma membrane tether formation force measurement by optical tweezers. Cell mechanical adhesiveness increased from 231 ± 149 Pa at 1 h to 1085 ± 211 Pa at 6 h. The cell contact area with the substrata increased from 161 ± 52 μm2 at 1 h to 369 ± 105 μm2 at 6 h. The tether formation force increased from 232 ± 23 pN at 1 h to 591 ± 17 pN at 6 h. Moreover, fluorescence staining by rhodamine‐phalloidin demonstrated the process of actin spreading within the cytoskeleton from 0.5 to 6 h and allowed for measurement of cell height which was found to decrease from 12.3 ± 2.9 μm at 0.5 h to 6.2 ± 0.9 μm at 6 h.

Methodology for estimation of time-dependent surface heat flux due to cryogen spray cooling

AbstractCryogen spray cooling (CSC) is an effective technique to protect the epidermis during cutaneous laser therapies. Spraying a cryogen onto the skin surface creates a time-varying heat flux, effectively cooling the skin during and following the cryogen spurt. In previous studies mathematical models were developed to predict the human skin temperature profiles during the cryogen spraying time. However, no studies have accounted for the additional cooling due to residual cryogen left on the skin surface following the spurt termination. We formulate and solve an inverse heat conduction (IHC) problem to predict the time-varying surface heat flux both during and following a cryogen spurt. The IHC formulation uses measured temperature profiles from within a medium to estimate the surface heat flux. We implement a one-dimensional sequential function specification method (SFSM) to estimate the surface heat flux from internal temperatures measured within an in vitro model in response to a cryogen spurt. Solution accuracy and experimental errors are examined using simulated temperature data. Heat flux following spurt termination appears substantial; however, it is less than that during the spraying time. The estimated time-varying heat flux can subsequently be used in forward heat conduction models to estimate temperature profiles in skin during and following a cryogen spurt and predict appropriate timing for onset of the laser pulse.

Epidermal protection with cryogen spray cooling during high fluence pulsed dye laser irradiation: An ex vivo study

Higher laser fluences than currently used in therapy (5–10 J/cm2) are expected to result in more effective treatment of port wine stain (PWS) birthmarks. However, higher incident fluences increase the risk of epidermal damage caused by absorption of light by melanin. Cryogen spray cooling offers an effective method to reduce epidermal injury during laser irradiation. The objective of this study was to determine whether high laser incident fluences (15–30 J/cm2) could be used while still protecting the epidermis in ex vivo human skin samples.

Internal temperature measurements in response to cryogen spray cooling of a skin phantom

Cryogen spray cooling (CSC) can protect the epidermis from non-specific thermal injury during laser treatment of port wine stains and other hypervascular cutaneous malformations. Knowledge of skin internal temperatures in response to CSC is essential for optimization of this technique. We used an epoxy resin compound to construct a kind phantom and measured its internal temperatures in response to cooling with different cryogens at various spurt durations, spraying distances, and ambient humidity levels. The measured temperature distributions during CSC were fitted by a mathematical model based on thermal diffusion theory. For spurt durations up to 100 ms, temperature reduction within the phantom remained confined to the upper 200 μm, and was affected by spraying distance. Depending on the cryogen used, temperature reductions up to 45°C could be measured 20 μm below the surface at the end of a 100 ms spurt. However, the cryogen film temperature on the epoxy resin surface was up to 35°C lower, indicating lack of perfect thermal contact at the cryogen film-phantom interface. Theoretical predictions were within 10% of measured temperatures. Ice formation occurred following termination of the spurt and was influenced by the ambient humidity level.

Regulation of pseudopodia localization in lymphocytes through application of mechanical forces by optical tweezers

T-lymphocytes are responsible for cell-mediated immunity, and recognize antigens on target cells (e.g., tumor cells, virus-infected cells) and antigen presenting cells (e.g., macrophages, dendritic cells). While mechanical forces applied to a cell surface can produce alterations in the cytoskeletal structure, leading to global structural rearrangements and changes in the intracellular biochemistry and gene expression, it remains unknown if local mechanical forces acting at the lymphocyte-antigen interaction site play any role in lymphocyte activation following antigen recognition. In this study we investigate the effect of such forces induced by optical tweezers on the lymphocytes morphological response. We brought optically trapped polystyrene beads, coated with a specific antibody against a clonotypic epitope of the T-cell receptors (TCRs), in contact with individual lymphocytes and applied mechanical forces at the TCR-antibody interaction site. Although bead size was a factor, simple bead contact tended to induce formation of pseudopodia that appeared randomly over the cells surface, while application of tangential forces at the interaction site redirected pseudopodia formation toward that site and promoted endocytosis activity. We propose that local forces play a key role in the initial lymphocyte adhesion to antigen-bearing cells, and may be implicated in antigen-specific motility, transendothelial migration, and tissue homing to sites of inflammation.

In-vivo study of epidermal protection by cryogen spray cooling during pulsed-laser irradiation at high radiant exposures

Cryogen spray cooling (CSC) offers a means to selectively cool the epidermis during laser therapy and has been used in conjunction with pulsed laser irradiation to treat light skin patients (Fitzpatrick Type I-TV) at moderate radiant exposures ( 15 J/cm2). Normal abdominal skin on twenty anesthetized individuals undergoing transverse rectus abdominis myocutaneous (TRAM) flap procedures with various skin types (Fitzpatrick type I-VT) were irradiated using incident radiant exposures of 8-30 J/cm2 without and with CSC. Assessment of tissue damage was based on histologic analysis. Epidermal damage was observed as basal epidermal cell vacuolization, epidermal basal layer separation, and epithelial cell spindling. For lighter skin patients (Fitzpatrick type IIV) the epidermal damage threshold was increased to as much as 30 J/cm2 when using CSC. However, complete epidermal protection in the darkest skin patients (Fitzpatrick type V-VT) could not be achieved with cryogen spurt durations as long as 300 ms using cunent CSC protocols.

Skin thermal response to sapphire contact and cryogen spray cooling : A comparative study based on measurements in a skin phantom

Non-specific thermal injury to the epidermis may occur as a result of laser treatment of cutaneous hypervascular malformations (e.g. port wine stains) and other dermatoses. Methods to protect the epidermis from thermal injury include sapphire contact cooling (SCC) and cryogen spray cooling (CSC). Evaluation of the skin thermal response to either cooling method and better understanding of the heat transfer process at the skin surface are essential for further optimization of cooling technique during laser therapy. We present internal temperature measurements in an epoxy resin phantom in response to both SCC and CSC, and use the results in conjunction with a mathematical model to predict the temperature distributions within human skin. Based on our results, a conductive heat transfer process at the skin interface appears to be the primary mechanism for both SCC and CSC. In the case of CSC, film cooling rather than evaporative cooling seems to be the dominant mode during the spurt duration. Currently, due to the lower temperature of the cryogen film and its shorter time of application, CSC produces larger temperature reductions at the skin surface and smaller temperature reductions at depths greater than 200 micrometer (i.e., higher spatial selectivity) when compared to SCC. However, SCC can potentially induce temperature reductions comparable to those produced by CSC if a sapphire temperature similar to that for a cryogen could be achieved in practice.

Thermal and fluid characteristics during cryogen spray cooling

Cryogen spray cooling (CSC) is a technique to protect the epidermis from non-specific thermal injury during laser treatment of various dermatoses. Successful application of CSC in conjunction with laser treatment of heavily pigmented individuals, and high radiant exposures which may be required for effective therapeutic outcomes, requires enhancement of heat removal. We have investigated the thermal mechanisms, and effects of droplet size, density and velocity on heat removal during CSC. Our results suggest that although the inherent thermal diffusivity of skin may be a limiting factor in heat removal, parameters such as droplet size, density, and velocity are important, and should be optimized for maximum heat removal.