Membrane Tether Formation from Outer Hair Cells with Optical Tweezers

Biophysical Journal. Mar, 2002  |  Pubmed ID: 11867454

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.

Methodology for Estimation of Time-dependent Surface Heat Flux Due to Cryogen Spray Cooling

Annals of Biomedical Engineering. Jan, 2002  |  Pubmed ID: 11874138

Cryogen 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.

Measurement of the Binding Forces Between Von Willebrand Factor and Variants of Platelet Glycoprotein Ibalpha Using Optical Tweezers

Lasers in Surgery and Medicine. 2002  |  Pubmed ID: 11948601

Thrombus formation is initiated by adhesion of the platelet receptor, glycoprotein (GP) Ib-IX-V complex, to its adhesive ligand, von Willebrand factor (vWf), in the subendothelium or plasma. The vWf-binding domain of GP Ib-IX-V is in the GP Ibalpha subunit of the complex and contains a leucine-rich repeat region. The adhesion of different leucine-rich repeats was studied using optical tweezers in order to determine which ones were critical for the vWf/GP Ibalpha interaction.

Ultralarge Multimers of Von Willebrand Factor Form Spontaneous High-strength Bonds with the Platelet Glycoprotein Ib-IX Complex: Studies Using Optical Tweezers

Blood. Jun, 2002  |  Pubmed ID: 12010796

Ultralarge von Willebrand factor (ULVWF) multimers have been implicated in the pathogenesis of the catastrophic microangiopathic disorder, thrombotic thrombocytopenic purpura. Spontaneous ULVWF binding to platelets has been ascribed to increased avidity due to the greatly increased number of binding sites for platelets (the A1 domain) per molecule. To address the mechanism of enhanced ULVWF binding to platelets, we used optical tweezers to study the unbinding forces from the glycoprotein Ib-IX (GP Ib-IX) complex of plasma VWF, ULVWF, and isolated A1 domain. The unbinding force was defined as the minimum force required to pull ligand-coated beads away from their attachment with GP Ib-IX-expressing cells. Beads coated with plasma VWF did not bind to the cells spontaneously, requiring the modulators ristocetin or botrocetin. The force required to break the ristocetin- and botrocetin-induced plasma VWF-GP Ib-IX bonds occurred in integer multiples of 6.5 pN and 8.8 pN, respectively, depending on the number of bonds formed. In contrast, beads coated with either ULVWF or A1 domain bound the cells in the absence of modulators, with bond strengths in integer multiples of approximately 11.4 pN for both. Thus, in the absence of shear stress, ULVWF multimers form spontaneous high-strength bonds with GP Ib-IX, while plasma VWF requires exogenous modulators. The strength of individual bonds formed with GP Ib-IX was similar for both ULVWF and the isolated A1 domain and greater than those of plasma VWF induced by either modulator. Therefore, we suggest that the conformational state of ULVWF multimers is more critical than their size for interaction with platelets.

Measurement of Adhesive Forces Between S. Epidermidis and Fibronectin-coated Surfaces Using Optical Tweezers

Lasers in Surgery and Medicine. 2002  |  Pubmed ID: 12124715

Biomaterial-mediated infection, a common cause of medical device failure, is initiated by bacterial adhesion to an adsorbed protein layer on the implant surface. This adhesion is thought to be mediated by specific molecules present on the bacterial cell surface. Optical tweezers can be used to measure the adhesive force between a single bacterium and a protein-coated surface.

Temporal Effects of Cell Adhesion on Mechanical Characteristics of the Single Chondrocyte

Journal of Orthopaedic Research : Official Publication of the Orthopaedic Research Society. Jan, 2003  |  Pubmed ID: 12507584

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 microm(2) at 1 h to 369+/-105 microm(2) 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 microm at 0.5 h to 6.2+/-0.9 microm at 6 h.

Cytoplasmic Truncation of Glycoprotein Ib Alpha Weakens Its Interaction with Von Willebrand Factor and Impairs Cell Adhesion

Biochemistry. Feb, 2003  |  Pubmed ID: 12590614

The interaction of the platelet glycoprotein (GP) Ib-IX-V complex with von Willebrand factor (VWF) is a critical step in the adhesion of platelets to the subendothelial matrix following endothelial cell damage, particularly under arterial flow conditions. In the human GP Ib-IX-V complex, the recognition of VWF appears to be mediated entirely by GP Ibalpha, the largest of four GP Ib-IX-V polypeptides. The goal of the present study was to investigate the involvement of the cytoplasmic domain of GP Ibalpha in the GP Ib-IX-VWF interaction under both static conditions and in the presence of high fluid shear stress. Using Chinese hamster ovary (CHO) cells that express GP Ibbeta, GP IX, and either wild-type GP Ibalpha or GP Ibalpha mutants missing various lengths of the cytoplasmic domain, we evaluated adhesion and flow-driven cell rolling on immobilized VWF in a parallel-plate flow chamber. Cells expressing GP Ibalpha polypeptides with truncations of 6-82 amino acids rolled faster than cells expressing wild-type GP Ibalpha. Cells that expressed polypeptides with intact actin-binding protein 280 binding sites (truncated to residue 582 of 610) rolled more slowly than those expressing GP Ibalpha with longer truncations. The rolling velocity of cells expressing truncated GP Ibalpha mutants increased with decreasing VWF coating density. In addition, a fraction of the truncated cells exhibited saltatory translocation at the lower VWF densities. Studies measuring the GP Ibalpha-VWF bond strength of three of the mutants using laser tweezers showed that progressive deletion of the cytoplasmic domain led to progressive weakening of the strength of individual GP Ibalpha-VWF bonds.

Measurement of Adhesive Forces Between Individual Staphylococcus Aureus MSCRAMMs and Protein-coated Surfaces by Use of Optical Tweezers

Journal of Bacteriology. Mar, 2003  |  Pubmed ID: 12618470

The force required to rupture bonds between individual Staphylococcus aureus MSCRAMMs and surfaces coated with extracellular matrix molecules has been quantified by using optical tweezers. The observed binding forces between fibrinogen or fibronectin and S. aureus MSCRAMMs occurred as an approximate integer multiple of 20 or 25 pN, respectively.

Optimum Pulse Duration and Radiant Exposure for Vascular Laser Therapy of Dark Port-wine Skin: a Theoretical Study

Applied Optics. Mar, 2003  |  Pubmed ID: 12638894

Laser therapy for cutaneous hypervascular malformations such as port-wine stain birthmarks is currently not feasible for dark-skinned individuals. We study the effects of pulse duration, radiant exposure, and cryogen spray cooling (CSC) on the thermal response of skin, using a Monte Carlo based optical-thermal model. Thermal injury to the epidermis decreases with increasing pulse duration during irradiation at a constant radiant exposure; however, maintaining vascular injury requires that the radiant exposure also increase. At short pulse durations, only a minimal increase in radiant exposure is necessary for a therapeutic effect to be achieved because thermal diffusion from the vessels is minimal. However, at longer pulse durations the radiant exposure must be greatly increased. There exists an optimum pulse duration at which minimal damage to the epidermis and significant injury within the targeted vasculature occur. For example, the model predicts optimum pulse durations of approximately 1.5, 6, and 20 ms for vessel diameters of 40, 80, and 120 microm, respectively. Optimization of laser pulse duration and radiant exposure in combination with CSC may offer a means to treat cutaneous lesions in dark-skinned individuals.

Methodology for Characterizing Heat Removal Mechanism in Human Skin During Cryogen Spray Cooling

Annals of Biomedical Engineering. May, 2003  |  Pubmed ID: 12757194

Cryogen spray cooling (CSC) reduces epidermal damage during laser treatment of various dermatoses. The goal of this study was to determine the heat removal mechanism in skin and quantify the amount in response to CSC. Thermocouples were imbedded in four model substrates with a range of thermal diffusivities, greater than three orders of magnitude in difference, to measure the temperature profiles in response to CSC and sapphire contact cooling, which removes heat completely by conduction. An algorithm solving an inverse heat conduction problem was subsequently used to quantify the amount of heat removal from the substrates using the measured temperatures. The interface thermal conductance and internal temperatures within the substrates were computed by a finite difference algorithm that solved the heat conduction equation. Results verify a marked increase in heat removal and interface thermal conductance with increasing thermal diffusivity. By estimation from the model substrate results, heat removal and interface thermal conductance values for skin were obtained. Data demonstrate that during CSC, evaporation is the dominant heat transfer mechanism in materials with higher thermal diffusivities; however, conductive cooling dominates in substrates with lower thermal diffusivities such as skin.

Effects of Cryogen Spray Cooling and High Radiant Exposures on Selective Vascular Injury During Laser Irradiation of Human Skin

Archives of Dermatology. Jun, 2003  |  Pubmed ID: 12810505

Increasing radiant exposure offers a means to increase treatment efficacy during laser-mediated treatment of vascular lesions, such as port-wine stains; however, excessive radiant exposure decreases selective vascular injury due to increased heat generation within the epidermis and collateral damage to perivascular collagen.

Thermal Response of Human Skin Epidermis to 595-nm Laser Irradiation at High Incident Dosages and Long Pulse Durations in Conjunction with Cryogen Spray Cooling: an Ex-vivo Study

Lasers in Surgery and Medicine. 2003  |  Pubmed ID: 12866117

Improved laser treatment of cutaneous hypervascular lesions is expected by utilizing higher incident dosages, longer pulse durations and longer wavelengths than those currently used in clinical settings. However, simply increasing the incident dosage will also increase the risk of nonspecific thermal injury to the epidermis due to light absorption by melanin. In this study, we investigated the thermal response of human skin epidermis to 595-nm wavelength laser irradiation at high incident dosages (up to 20 J/cm(2)) and long pulse durations (up to 40 milliseconds) in conjunction with cryogen spray cooling (CSC) using ex-vivo human skin samples.

Quantification of S. Aureus Binding to Extracellular Matrix Molecules Using Optical Tweezers

IEEE Engineering in Medicine and Biology Magazine : the Quarterly Magazine of the Engineering in Medicine & Biology Society. Sep-Oct, 2003  |  Pubmed ID: 14699944

Dynamic Measurements of Transverse Optical Trapping Force in Biological Applications

Annals of Biomedical Engineering. Jul, 2004  |  Pubmed ID: 15298439

Optical tweezers present a technology for measurements of biological forces in the piconewton range. In such applications, one method of calibrating the transverse optical trapping force involves relating a known external force to the displacement of the trapped object from the trapping center. In this work we used Fourier analysis of the equation of motion to calculate the displacement of the trapped object from the trapping center under an external force induced by viscous drag. Triangular waveforms of different frequencies were used both in theoretical modeling and experiments to induce a force on a trapped object. We investigated the contribution of various factors including frequency of the external force, fluid viscosity, density, and dimensions of the trapped object, stiffness of the optical trap, and frequency response of the instruments used to control the motion of the viscous medium to the accuracy of the calibration. The developed model can be adopted for calibration of the transverse trapping force, analysis of the trapped object motion, and reconstruction of a force profile during measurements of dynamic biological forces.

Laser Irradiation of the Guinea Pig Basilar Membrane

Lasers in Surgery and Medicine. 2004  |  Pubmed ID: 15389736

The cochlea is the part of the inner ear that transduces sound waves into neural signals. The basilar membrane, a connective tissue sheet within the cochlea, is tonotopically tuned based on the spatial variation of its mass, stiffness, and damping. These biophysical properties are mainly defined by its constituent collagen fibers. We sought to assess the effect of laser irradiation on collagen within the basilar membrane using histological analysis.

Effects of Droplet Velocity, Diameter, and Film Height on Heat Removal During Cryogen Spray Cooling

Annals of Biomedical Engineering. Aug, 2004  |  Pubmed ID: 15446509

Cryogen spray cooling (CSC) is an effective method to reduce or eliminate epidermal damage during laser treatment of various dermatoses. This study sought to determine the effects of specific cryogen properties on heat removal. Heat removal was quantified using an algorithm that solved an inverse heat conduction problem from internal temperature measurements made within a skin phantom. A nondimensional parameter, the Weber number, characterized the combined effects of droplet velocity, diameter, and surface tension. CSC experiments with laser irradiation were conducted on ex vivo human skin samples to assess the effect of Weber number on epidermal protection. An empirical relationship between heat removal and the difference in droplet temperature and the substrate, droplet velocity, and diameter was obtained. Histological sections of irradiated ex vivo human skin demonstrated that sprays with higher Weber numbers increased epidermal protection. Results indicate that the cryogen film acts as an impediment to heat transfer between the impinging droplets and the substrate. This study offers the importance of Weber number in heat removal and epidermal protection.

Regulation of Pseudopodia Localization in Lymphocytes Through Application of Mechanical Forces by Optical Tweezers

Journal of Biomedical Optics. Sep-Oct, 2004  |  Pubmed ID: 15447007

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 lymphocyte's 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 cell's 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.

Adherence of Staphylococcus Aureus Fibronectin Binding Protein A Mutants: an Investigation Using Optical Tweezers

Biomolecular Engineering. Nov, 2004  |  Pubmed ID: 15567104

Bacterial adhesion to extracellular matrix proteins plays a major role in infections of host tissue and medical devices. In some species of gram-positive cocci, this adhesion is mediated by specific molecules present on the bacterial cell surface. We have used optical tweezers to dynamically measure the adhesive force between an individual Staphylococcus aureus bacterium and a fibronectin-coated surface. A bacterium was optically trapped and brought in contact with a 10-microm diameter polystyrene microsphere coated with fibronectin. The force required to detach the cell from the microsphere was measured by tracking the displacement signals of the trapped cell on a quadrant photodiode throughout the detachment process for a series of S. aureus strains expressing fibronectin-binding proteins with various degrees of mutation. The single-bond rupture forces ranged between 15 and 26 pN depending on the extent of mutation. No binding was observed in the strain with the highest degree of mutation. These results confirm that multiple regions of the S. aureus fibronectin adhesin participate in the binding process and provide further insight into the role of these regions in the adhesive process.

Comparison of Human Skin Opto-thermal Response to Near-infrared and Visible Laser Irradiations: a Theoretical Investigation

Physics in Medicine and Biology. Nov, 2004  |  Pubmed ID: 15584524

Near-infrared wavelengths are absorbed less by epidermal melanin, and penetrate deeper into human skin dermis and blood than visible wavelengths. Therefore, laser irradiation using near-infrared wavelengths may improve the therapeutic outcome of cutaneous hyper-vascular malformations in moderately to heavily pigmented skin patients and those with large-sized blood vessels or blood vessels extending deeply into the skin. A mathematical model composed of a Monte Carlo algorithm to estimate the distribution of absorbed light, numerical solution of a bio-heat diffusion equation to calculate the transient temperature distribution, and a damage integral based on an empirical Arrhenius relationship to quantify the tissue damage was utilized to investigate the optothermal response of human skin to near-infrared and visible laser irradiations in conjunction with cryogen spray cooling. In addition, the thermal effects of a single continuous laser pulse and micropulse-composed laser pulse profiles were compared. Simulation results indicated that a 940 nm wavelength induces improved therapeutic outcome compared with a 585 and 595 nm wavelengths for the treatment of patients with large-sized blood vessels and moderately to heavily pigmented skin. On the other hand, a 585 nm wavelength shows the best efficacy in treating small-sized blood vessels, as characterized by the largest laser-induced blood vessel damage depth compared with 595 and 940 nm wavelengths. Dermal blood content has a considerable effect on the threshold incident dosage for epidermal damage, while the effect of blood vessel size is minimal. For the same macropulse duration and incident dosage, a micropulse-composed pulse profile results in higher peak temperature at the basal layer of skin epidermis than an ideal single continuous pulse profile.

P-selectin Anchors Newly Released Ultralarge Von Willebrand Factor Multimers to the Endothelial Cell Surface

Blood. Mar, 2004  |  Pubmed ID: 14630802

von Willebrand factor (VWF) released from endothelium is ultralarge (UL) and hyperreactive. If released directly into plasma, it can spontaneously aggregate platelets, resulting in systemic thrombosis. This disastrous consequence is prevented by the ADAMTS13 (ADisintegrin and Metalloprotease with ThromboSpondin motif) cleavage of ULVWF into smaller, less active forms. We previously showed that ULVWF, on release, forms extremely long stringlike structures. ADAMTS13 cleaves these strings under flow significantly faster than it does under static conditions. As ULVWF tethering to endothelium is important for its rapid proteolysis, we investigated 2 molecules for their potential to anchor the ULVWF strings: P-selectin and integrin alpha v beta 3. We demonstrated that P-selectin anchors ULVWF to endothelium by several means. First, Chinese hamster ovary (CHO) cells expressing P-selectin specifically adhered to immobilized ULVWF and ULVWF-coated beads to immobilized P-selectin. Second, an anti-VWF antibody coimmunoprecipitates P-selectin from the histamine-activated endothelial cells. Third, P-selectin antibody or soluble P-selectin, but not a alpha v beta 3 antibody, RGDS peptide, or heparin, blocked the formation of ULVWF strings. Fourth, P-selectin expression was in clusters predominantly along the ULVWF strings. Finally, the strength of the minimal ULVWF-P-selectin bond was measured to be 7.2 pN. We, therefore, conclude that P-selectin may anchor ULVWF strings to endothelial cells and facilitate their cleavage by ADAMTS13.

Combining Optical Tweezers and Patch Clamp for Studies of Cell Membrane Electromechanics

The Review of Scientific Instruments. Sep, 2004  |  Pubmed ID: 21412445

We have designed and implemented a novel experimental setup which combines optical tweezers with patch-clamp apparatus to investigate the electromechanical properties of cellular plasma membranes. In this system, optical tweezers provide measurement of forces at piconewton scale, and the patch-clamp technique allows control of the cell transmembrane potential. A micron-size bead trapped by the optical tweezers is brought in contact with the membrane of a voltage-clamped cell, and subsequently moved away to form a plasma membrane tether. Bead displacement from the trapping center is monitored by a quadrant photodetector for dynamic measurements of tether force. Fluorescent beads and the corresponding fluorescence imaging optics are used to eliminate the shadow of the cell projected on the quadrant photodetector. Salient information associated with the mechanical properties of the membrane tether can thus be obtained. A unique feature of this setup is that the patch-clamp headstage and the manipulator for the recording pipette are mounted on a piezoelectric stage, preventing relative movements between the cell and the patch pipette during the process of tether pulling. Tethers can be pulled from the cell membrane at different holding potentials, and the tether force response can be measured while changing transmembrane potential. Experimental results from mammalian cochlear outer hair cells and human embryonic kidney cells are presented.

High Frequency Electrically-induced Force Generation by Cellular Plasma Membranes

Conference Proceedings : ... Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Conference. 2005  |  Pubmed ID: 17282369

Using a novel experimental technique that combines optical trapping with patch-clamp and fluorescence photometry, we provide preliminary evidence that native biological membranes are capable of electrically-induced piconewton level force generation in the absence of specialized transmembrane proteins. Force generation is dependent on membrane tension and the transmembrane electrical potential. Salicylate diminishes and presence of prestin, a transmembrane protein found in cochlear outer hair cells, enhances force generation.

Comparison of 585 and 595 Nm Laser-induced Vascular Response of Normal in Vivo Human Skin

Lasers in Surgery and Medicine. Feb, 2005  |  Pubmed ID: 15704165

Two wavelengths, 585 and 595 nm, are currently common options for treating vascular malformations such as port-wine stains (PWS). Controversy exists as to which wavelength induces greater photothermal damage to the blood vessels and subsequent resolution of the malformations.

Dynamic Force Spectroscopy of Glycoprotein Ib-IX and Von Willebrand Factor

Biophysical Journal. Jun, 2005  |  Pubmed ID: 15764659

The first stage in hemostasis is the binding of the platelet membrane receptor glycoprotein (GP) Ib-IX complex to the A1 domain of von Willebrand factor in the subendothelium. A bleeding disorder associated with this interaction is platelet-type von Willebrand disease, which results from gain-of-function (GOF) mutations in amino acid residues 233 or 239 of the GP Ibalpha subunit of GP Ib-IX. Using optical tweezers and a quadrant photodetector, we investigated the binding of A1 to GOF and loss-of-function mutants of GP Ibalpha with mutations in the region containing the two known naturally occurring mutations. By dynamically measuring unbinding force profiles at loading rates ranging from 200-20,000 pN/s, we found that the bond strengths between A1 and GP Ibalpha GOF mutants (233, 235, 237, and 239) were significantly greater than the A1/wild-type GP Ib-IX bond at all loading rates examined (p < 0.05). In addition, mutants 231 and 232 exhibited significantly lower bond strengths with A1 than the wild-type receptors (p < 0.05). We computed unloaded dissociation rate constant (k(off)(0)) values for interactions involving mutant and wild-type GP Ib-IX receptors with A1 and found the A1/wild-type GP Ib-IX k(off)(0) value of 5.47 +/- 0.25 s(-1) to be significantly greater than the GOF k(off)(0) values and significantly less than the loss-of-function k(off)(0) values. Our data illustrate the importance of the bond kinetics associated with the VWF/GP Ib-IX interaction in hemostasis and also demonstrate the drastic changes in binding that can occur when only a single amino acid of GP Ibalpha is altered.

Effects of Salicylate on Plasma Membrane Mechanics

Journal of Neurophysiology. Sep, 2005  |  Pubmed ID: 15958599

High concentrations of the amphipathic drug salicylate (Sal) block outer hair cell (OHC) electromotility resulting in reversible hearing loss. We used optical tweezers to study the effects of Sal on the mechanics of the cell plasma membrane. Membrane tethers were formed from guinea pig OHCs and cultured human embryonic kidney (HEK) cells as controls. HEK cells are commonly used in functional expression studies of electromotility. Effective tether viscosity (eta(eff)), steady-state tethering force extrapolated to zero pulling rate F(ss0), and time constant for tether growth (tau(tg)) were estimated from the measurements of the instantaneous tethering force at different tether pulling rates. Average values of eta(eff), F(ss0), and tau(tg) for the OHC lateral wall plasma membrane and control cell plasma membrane remained the same after Sal perfusion, which is consistent with the hypothesis that Sal-induced reversible hearing loss appears to be more the result of its competition with essential anions and less the result of a change in plasma membrane mechanics.

Epidermal and Vascular Damage Analysis of in Vivo Human Skin in Response to 595 Nm Pulsed Laser Irradiation

Lasers in Surgery and Medicine. Jul, 2005  |  Pubmed ID: 15971245

Laser irradiation is the current modality for treatment of cutaneous hypervascular malformations such as port wine stains and telangiectasia. Although cryogen spray cooling (CSC) is used to protect the epidermis from non-specific laser-induced thermal damage in moderately-pigmented skin types, individuals with high melanin content are still at risk for epidermal damage using the current laser irradiation and CSC parameters. The objective of this study was to investigate the influence of the spray Weber number (1,100 or 5,100) on epidermal protection and examine vascular coagulation in response to pulsed dye laser irradiation.

Laser-induced Thermal Injury to Dermal Blood Vessels: Analysis of Wavelength (585 Nm Vs. 595 Nm), Cryogen Spray Cooling, and Wound Healing Effects

Lasers in Surgery and Medicine. Sep, 2005  |  Pubmed ID: 16175632

Successful laser treatment of cutaneous hyper-vascular lesions requires appropriate laser irradiation parameters for selective photothermolysis of ectatic dermal blood vessels as well as appropriate cooling parameters for epidermal protection based on an individual patient basis. Using the rabbit ear as an in vivo model for dermal vasculature, we investigated the influences of laser wavelength (585 nm vs. 595 nm) and cryogen spray cooling with various spurt durations on the laser-induced thermal injury to dermal blood vessels. Wound healing response was also evaluated in 2 hours and 4 days.

Effects of Chlorpromazine on Mechanical Properties of the Outer Hair Cell Plasma Membrane

Biophysical Journal. Dec, 2005  |  Pubmed ID: 16199506

An optical tweezers system was used to characterize the effects of chlorpromazine (CPZ) on the mechanical properties of the mammalian outer hair cell (OHC) through the formation of plasma membrane tethers. Such tethers exhibited force relaxation when held at a constant length for several minutes. We used a second-order generalized Kelvin body to model tether-force behavior from which several mechanical parameters were then calculated including stiffness, viscosity-associated measures, and force relaxation time constants. The results of the analysis portray a two-part relaxation process characterized by significantly different rates of force decay, which we propose is due to the local reorganization of lipids within the tether and the flow of external lipid into the tether. We found that CPZ's effect was limited to the latter phenomenon since only the second phase of relaxation was significantly affected by the drug. This finding coupled with an observed large reduction in overall tether forces implies a common basis for the drug's effects, the plasma membrane-cytoskeleton interaction. The CPZ-induced changes in tether viscoelastic behavior suggest that alterations in the mechanical properties of the OHC lateral wall could play a role in the modulation of OHC electromotility by CPZ.

Autofluorescence Characterization for the Early Diagnosis of Neoplastic Changes in DMBA/TPA-induced Mouse Skin Carcinogenesis

Lasers in Surgery and Medicine. Dec, 2005  |  Pubmed ID: 16240416

Squamous cell carcinoma (SCC), the second most common skin cancer, usually remains confined to the epidermis for some time but eventually penetrates the underlying tissues, if left untreated. The non-invasive early detection of the SCC is important for appropriate therapeutic strategies. In this study, we aim to characterize the tissue transformation in DMBA/TPA induced mouse skin tumor model using autofluorescence excitation emission matrix (EEM) in conjunction with a multivariate statistical method for early detection of the neoplastic changes.

Synchronous Fluorescence Spectroscopic Characterization of DMBA-TPA-induced Squamous Cell Carcinoma in Mice

Journal of Biomedical Optics. Jan-Feb, 2006  |  Pubmed ID: 16526889

While initially confined to the epidermis, squamous cell carcinoma can eventually penetrate into the underlying tissue if not diagnosed early and treated. The noninvasive early detection of the carcinoma is important to achieve a complete treatment of the disease. Of the various non-invasive optical techniques, the synchronous fluorescence (SF) technique is considered to provide a simplified spectral profile with more sharp spectral signatures of the endogenous fluorophores in complex systems. The potential use of the SF technique in the characterization of the sequential tissue transformation in 7,12-dimethylbenz(a)anthracene-12-O-tetradecanoylphorbol-13-acetate (DMBA-TPA)-induced mouse skin tumor model in conjunction with simple statistical analysis is explored. The SF spectra show distinct differences during the earlier weeks of the tumor-induction period. Intensity ratio variables are calculated and used in three discriminant analyses. All the discriminant analyses show better classification results with accuracy greater than 80%. From the observed differences in the spectral characteristics and the ratio variables that resulted in better classification between groups, it is concluded that tryptophan, collagen, and NADH are the key fluorophores that undergo changes during tissue transformation process and hence they can be targeted as tumor markers to diagnose normal from abnormal tissues using the SF technique.

Comparative Study of Cryogen Spray Cooling with R-134a and R-404a: Implications for Laser Treatment of Dark Human Skin

Journal of Biomedical Optics. Jul-Aug, 2006  |  Pubmed ID: 16965144

Cutaneous laser treatment in dark skin patients is challenging due to significant light absorption by the melanin at the basal layer of epidermis, which can result in irreversible nonspecific thermal injury to the epidermis. Cryogen spray cooling (CSC) with R-134a (boiling point approximately -26.2 degrees C at 1 atm), which is currently used during cutaneous laser treatment, has shown poor efficacy in protecting dark human skin. We investigated the potential of CSC with R-404a (boiling point approximately -46.5 degrees C at 1 atm), which has a lower boiling point than R-134a, for improved therapeutic outcome in dark human skin at three levels: in vitro (epoxy resin skin phantom), ex vivo (normal dark human skin sample), and in vivo (skin of the rabbit external ear). The skin phantom was used to acquire the surface and internal temperature profiles in response to CSC with R-134a or R-404a at various spurt durations, based upon which CSC-induced heat removal from the skin phantom was estimated using an algorithm that solved a one-dimensional inverse heat conduction problem. CSC with R-404a increased the temperature reductions within the phantom and subsequently the amount of heat removal from the phantom in comparison to that with R-134a. Normal ex vivo Fitzpatrick types V-VI human skin samples were used to investigate the thermal response of dark human skin epidermis to CSC (R-134a or R-404a) at various spurt durations in conjunction with 595-nm pulsed dye laser irradiation at various radiant exposures. Cryogen R-404a increased the threshold radiant exposures for irreversible thermal injury to the epidermis in dark pigmentation skin. No obvious CSC-induced morphological changes to human skin was observed when sprayed with R404-a spurts using durations up to 300 ms. In vivo rabbit ear vasculature was used as a model of cutaneous anomalies to assess the influences of CSC (with R-134a or R-404a) on the photothermolysis of dermal blood vessels. CSC (R-134a or R-404a) with the spurt durations of 100 to 300 ms increased the most superficial depth of thermally damaged dermal blood vessel compared with the sites without CSC, implying possible nonspecific cooling of superficial dermal blood vessels by the cryogen spurts with the settings applied.

Standing Wave Total Internal Reflection Fluorescence Microscopy to Measure the Size of Nanostructures in Living Cells

Journal of Biomedical Optics. Nov-Dec, 2006  |  Pubmed ID: 17212536

We present the first application of standing wave fluorescence microscopy (SWFM) to determine the size of biological nanostructures in living cells. The improved lateral resolution of less than 100 nm enables superior quantification of the size of subcellular structures. We demonstrate the ability of SWFM by measuring the diameter of biological nanotubes (membrane tethers formed between cells). The combination of SWFM with total internal reflection (TIR), referred to as SW-TIRFM, allows additional improvement of axial resolution by selective excitation of fluorescence in a layer of about 100 nm.

Prestin Modulates Mechanics and Electromechanical Force of the Plasma Membrane

Biophysical Journal. Jul, 2007  |  Pubmed ID: 17468166

The voltage-dependent movement, or electromotility, of cochlear outer hair cells contributes to cochlear amplification in mammalian hearing. Outer hair-cell electromotility involves a membrane-based motor in which the membrane protein prestin plays a central role. We have investigated the contribution of prestin to the mechanics and electromechanical force (EMF) generation of the membrane using membrane tethers formed from human embryonic kidney (HEK) cells. Several measures of membrane tether mechanics are greater in tethers pulled from HEK cells transfected with prestin when compared to control untransfected HEK cells. A single point mutation of alanine to tryptophan (A100W) in prestin eliminates prestin-associated charge movement and diminishes EMF but does not alter passive membrane mechanics. These results suggest that prestin-associated charge transfer is necessary for maximal EMF generation by the membrane.

Laser-induced Collagen Remodeling and Deposition Within the Basilar Membrane of the Mouse Cochlea

Journal of Biomedical Optics. Mar-Apr, 2007  |  Pubmed ID: 17477714

The cochlea is the mammalian organ of hearing. Its predominant vibratory element, the basilar membrane, is tonotopically tuned, based on the spatial variation of its mass and stiffness. The constituent collagen fibers of the basilar membrane affect its stiffness. Laser irradiation can induce collagen remodeling and deposition in various tissues. We tested whether similar effects could be induced within the basilar membrane. Trypan blue was perfused into the scala tympani of anesthetized mice to stain the basilar membrane. We then irradiated the cochleas with a 694-nm pulsed ruby laser at 15 or 180 Jcm(2). The mice were sacrificed 14 to 16 days later and collagen organization was studied. Polarization microscopy revealed that laser irradiation increased the birefringence within the basilar membrane in a dose-dependent manner. Electron microscopy demonstrated an increase in the density of collagen fibers and the deposition of new fibrils between collagen fibers after laser irradiation. As an assessment of hearing, auditory brainstem response (ABR) thresholds were found to increase moderately after 15 Jcm(2) and substantially after 180 Jcm(2). Our results demonstrate that collagen remodeling and new collagen deposition occurs within the basilar membrane after laser irradiation in a similar fashion to that found in other tissues.

Laser-induced Heating of Dextran-coated Mesocapsules Containing Indocyanine Green

Biotechnology Progress. Nov-Dec, 2007  |  Pubmed ID: 17914861

Indocyanine green (ICG) is a photosensitive reagent with clinically relevant diagnostic and therapeutic applications. Recently, ICG has been investigated for its utility as an exogenous chromophore during laser-induced heating. However, ICG's effectiveness remains hindered by its molecular instability, rapid circulation kinetics, and nonspecific systemic distribution. To overcome these limitations, we have encapsulated ICG within dextran-coated mesocapsules (MCs). Our objective in this study was to explore the ability of MCs to induce thermal damage in response to laser irradiation. To simulate tumorous tissue targeted with MCs, cylindrical phantoms were prepared consisting of gelatin, intralipid emulsion, and various concentrations of MCs. The phantoms were embedded within fresh chicken breast tissue representing surrounding normal tissue. The tissue models were irradiated at lambda = 808 nm for 10 min at constant power (P = 4.2 W). Five hypodermic thermocouples were used to record the temperature at various depths below the tissue surface and transverse distances from the laser beam central axis during irradiation. Temperature profiles were processed to remove the baseline temperature and influence of light absorption by the thermocouple and subsequently used to calculate a damage index based on the Arrhenius damage integral. Tissue models containing MCs experienced a maximum temperature change of 18.5 degrees C. Damage index calculations showed that the heat generation from MCs at these parameters is sufficient to induce thermal damage, while no damage was predicted in the absence of MCs. ICG maintains its heat-generating capabilities in response to NIR laser irradiation when encapsulated within MCs. Such encapsulation provides a potentially useful methodology for laser-induced therapeutic strategies.

Effects of Prestin on Membrane Mechanics and Electromechanics

Conference Proceedings : ... Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Conference. 2007  |  Pubmed ID: 18003225

The electromotility of cochlear outer hair cells (OHCs) is a required process for normal hearing, and involves a membrane-based mechanism in which the transmembrane protein, prestin, plays a central role. We have investigated the contribution of prestin to the mechanics and electromechanics of the cell membrane using membrane tethers formed from human embryonic kidney cells. Our results suggest that prestin appears to change membrane tension and amplify electrically-evoked force generation, while a single point mutation of alanine to tryptophan in prestin reduces electrically-evoked force generation without affecting the membrane tension. We propose that prestin and membrane work in synergy to produce the electrical and mechanical changes that are required during OHC electromotility.

Stability Assessment of Indocyanine Green Within Dextran-coated Mesocapsules by Absorbance Spectroscopy

Journal of Biomedical Optics. Nov-Dec, 2007  |  Pubmed ID: 18163847

The biocompatibility and high absorption in the near IR range of indocyanine green (ICG) have made it a suitable candidate chromophore for optical imaging and laser-mediated therapy of superficial tumors. However, its clinical efficacy remains limited by factors such as rapid circulation kinetics, lack of target specificity, and molecular instability. Such drawbacks motivated us to encapsulate ICG into carrier particles to improve target specificity and retention time. We use absorbance spectroscopy to investigate the effects of encapsulating ICG within dextran-coated capsules. The mesocapsules (MCs) containing ICG are synthesized using a previously reported charge-assembly technique. Both freely dissolved ICG and ICG-MCs are prepared with ICG concentrations of either 50 or 10 microg/ml. Samples are exposed either to a broadband light source or incubated at 3, 23, or 40 degrees C. Absorbance spectra are recorded at various time points up to 96 h. At the lower concentration of 10 microg/ml, ICG within MCs experiences less light-induced degradation. The MC system also protects ICG from thermal degradation at all tested temperatures. The polymer-salt aggregate core of the MCs hinders the mobility of ICG molecules. The MC system shields ICG from vibrational and translational agitation as well as molecular changes such as fragmentation.

Studies of Plasma Membrane Mechanics and Plasma Membrane-cytoskeleton Interactions Using Optical Tweezers and Fluorescence Imaging

Journal of Biomechanics. 2007  |  Pubmed ID: 16500663

We use optical tweezers in conjunction with an optical position-sensing system, which spectrally filters signals generated by a trapped fluorescent microsphere to study plasma membrane (PM) mechanics and its interactions with cytoskeleton. We dynamically measure the PM tethering force on human embryonic kidney cells that are a standard cultured cell line. Recorded tethering force vs. PM displacement profiles, revealed the tether formation process, initiated with linear deformation of the PM, followed by a nonlinear regime and terminated with the local separation of PM. Tethering force vs. displacement profiles were used to estimate tether formation force and stiffness parameter of the PM. Integration of the force-displacement profiles yielded the work of tether formation, including linear and nonlinear components. Our results demonstrate that spectral filtering of the optically trapped fluorescent microsphere image formed on the position-sensing system overcomes the artifacts introduced by the transillumination imaging and allows accurate measures of PM mechanics before and during the initial stages of tether formation.

Modeling the Mechanics of Tethers Pulled from the Cochlear Outer Hair Cell Membrane

Journal of Biomechanical Engineering. Jun, 2008  |  Pubmed ID: 18532856

Cell membrane tethers are formed naturally (e.g., in leukocyte rolling) and experimentally to probe membrane properties. In cochlear outer hair cells, the plasma membrane is part of the trilayer lateral wall, where the membrane is attached to the cytoskeleton by a system of radial pillars. The mechanics of these cells is important to the sound amplification and frequency selectivity of the ear. We present a modeling study to simulate the membrane deflection, bending, and interaction with the cytoskeleton in the outer hair cell tether pulling experiment. In our analysis, three regions of the membrane are considered: the body of a cylindrical tether, the area where the membrane is attached and interacts with the cytoskeleton, and the transition region between the two. By using a computational method, we found the shape of the membrane in all three regions over a range of tether lengths and forces observed in experiments. We also analyze the effects of biophysical properties of the membrane, including the bending modulus and the forces of the membrane adhesion to the cytoskeleton. The model's results provide a better understanding of the mechanics of tethers pulled from cell membranes.

In-vivo Fluorescence Imaging of Mammalian Organs Using Charge-assembled Mesocapsule Constructs Containing Indocyanine Green

Optics Express. Dec, 2008  |  Pubmed ID: 19065196

Indocyanine green (ICG) is a fluorescent probe used in clinical imaging. However, its utility remains limited by optical instability, rapid circulation kinetics, and exclusive uptake by the liver. Using mesocapsule (MC) constructs to encapsulate ICG, we have developed a technology to stabilize ICG's optical properties and alter its biodistribution. We present in vivo fluorescence images of mammalian organs to demonstrate the potential application of our ICG encapsulation technology for optical imaging of specific tissues.

Biodistribution of Encapsulated Indocyanine Green in Healthy Mice

Molecular Pharmaceutics. Sep-Oct, 2009  |  Pubmed ID: 19799463

Indocyanine green (ICG) is a fluorescent probe used in various optically mediated diagnostic and therapeutic applications. However, utility of ICG remains limited by its unstable optical properties and nonspecific localization. We have encapsulated ICG within electrostatically assembled mesocapsules (MCs) to explore its potential for targeted optical imaging and therapy. In this study, we investigate how the surface coating and size of the MCs influences ICG's biodistribution in vivo. ICG was administered intravenously to Swiss Webster mice as a free solution or encapsulated within either 100 nm diameter MCs coated with dextran; 500 nm diameter MCs coated with dextran; or 100 nm diameter MCs coated with 10 nm ferromagnetic iron oxide nanoparticles, themselves coated with polyethylene glycol. ICG was extracted from harvested blood and organs at various times and its amount quantified with fluorescence measurements. MCs containing ICG accumulated in organs of the reticuloendothelial system, namely, the liver and spleen, as well as the lungs. The circulation kinetics of ICG appeared unaffected by encapsulation; however, the deposition within organs other than the liver suggests a different biodistribution mechanism. Results suggest that the capsules' coating influences their biodistribution to a greater extent than their size. The MC encapsulation system allows for delivery of ICG to organs other than the liver, enabling the potential development of new optical imaging and therapeutic strategies.

Computational Analysis of the Tether-pulling Experiment to Probe Plasma Membrane-cytoskeleton Interaction in Cells

Physical Review. E, Statistical, Nonlinear, and Soft Matter Physics. Oct, 2009  |  Pubmed ID: 19905340

Tethers are thin membrane tubes that can be formed when relatively small and localized forces are applied to cellular membranes and lipid bilayers. Tether pulling experiments have been used to better understand the fine membrane properties. These include the interaction between the plasma membrane and the underlying cytoskeleton, which is an important factor affecting membrane mechanics. We use a computational method aimed at the interpretation and design of tether pulling experiments in cells with a strong membrane-cytoskeleton attachment. In our model, we take into account the detailed information in the topology of bonds connecting the plasma membrane and the cytoskeleton. We compute the force-dependent piecewise membrane deflection and bending as well as modes of stored energy in three major regions of the system: body of the tether, membrane-cytoskeleton attachment zone, and the transition zone between the two. We apply our method to three cells: cochlear outer hair cells (OHCs), human embryonic kidney (HEK) cells, and Chinese hamster ovary (CHO) cells. OHCs have a special system of pillars connecting the membrane and the cytoskeleton, and HEK and CHO cells have the membrane-cytoskeleton adhesion arrangement via bonds (e.g., PIP2), which is common to many other cells. We also present a validation of our model by using experimental data on CHO and HEK cells. The proposed method can be an effective tool in the analyses of experiments to probe the properties of cellular membranes.

Self-assembly Synthesis, Tumor Cell Targeting, and Photothermal Capabilities of Antibody-coated Indocyanine Green Nanocapsules

Journal of the American Chemical Society. Feb, 2010  |  Pubmed ID: 20092330

New colloidal materials that can generate heat upon irradiation are being explored for photothermal therapy as a minimally invasive approach to cancer treatment. The near-infrared dye indocyanine green (ICG) could serve as a basis for such a material, but its encapsulation and subsequent use are difficult to carry out. We report the three-step room-temperature synthesis of approximately 120-nm capsules loaded with ICG within salt-cross-linked polyallylamine aggregates, and coated with antiepidermal growth factor receptor (anti-EGFR) antibodies for tumor cell targeting capability. We studied the synthesis conditions such as temperature and water dilution to control the capsule size and characterized the size distribution via dynamic light scattering and scanning electron microscopy. We further studied the specificity of tumor cell targeting using three carcinoma cell lines with different levels of EGFR expression and investigated the photothermal effects of ICG containing nanocapsules on EGFR-rich tumor cells. Significant thermal toxicity was observed for encapsulated ICG as compared to free ICG at 808 nm laser irradiation with radiant exposure of 6 W/cm(2). These results illustrate the ability to design a colloidal material with cell targeting and heat generating capabilities using noncovalent chemistry.

Cell Membrane Tethers Generate Mechanical Force in Response to Electrical Stimulation

Biophysical Journal. Aug, 2010  |  Pubmed ID: 20682262

Living cells maintain a huge transmembrane electric field across their membranes. This electric field exerts a force on the membrane because the membrane surfaces are highly charged. We have measured electromechanical force generation by cell membranes using optically trapped beads to detach the plasma membrane from the cytoskeleton and form long thin cylinders (tethers). Hyperpolarizing potentials increased and depolarizing potentials decreased the force required to pull a tether. The membrane tether force in response to sinusoidal voltage signals was a function of holding potential, tether diameter, and tether length. Membrane electromechanical force production can occur at speeds exceeding those of ATP-based protein motors. By harnessing the energy in the transmembrane electric field, cell membranes may contribute to processes as diverse as outer hair cell electromotility, ion channel gating, and transport.

Optical Nano-constructs Composed of Genome-depleted Brome Mosaic Virus Doped with a Near Infrared Chromophore for Potential Biomedical Applications

ACS Nano. Feb, 2011  |  Pubmed ID: 21210643

We have engineered an optical nanoconstruct composed of genome-depleted brome mosaic virus doped with indocyanine green (ICG), an FDA-approved near-infrared (NIR) chromophore. Constructs are highly monodispersed with standard deviation of ±3.8 nm from a mean diameter of 24.3 nm. They are physically stable and exhibit a high degree of optical stability at physiological temperature (37 °C). Using human bronchial epithelial cells, we demonstrate the effectiveness of the constructs for intracellular optical imaging in vitro, with greater than 90% cell viability after 3 h of incubation. These constructs may serve as a potentially nontoxic and multifunctional nanoplatform for site-specific deep-tissue optical imaging, and therapy of disease.

Special Section Guest Editorial: Photonics and Nanotechnology in Biophysics and Biomedical Research

Journal of Biomedical Optics. May, 2011  |  Pubmed ID: 21639561

Effect of Polyethylene Glycol Coatings on Uptake of Indocyanine Green Loaded Nanocapsules by Human Spleen Macrophages in Vitro

Journal of Biomedical Optics. May, 2011  |  Pubmed ID: 21639563

Near-infrared (NIR) optically active nanoparticles are promising exogenous chromophores for applications in medical imaging and phototherapy. Since nanoparticles can be rapidly eliminated from the body by cells of the reticuloendothelial system, a thriving strategy to increase their blood circulation time is through surface modification with polyethylene glycol (PEG). We constructed polymeric nanocapsules loaded with indocyanine green (ICG), an FDA-approved NIR dye, and coated with aldehyde-terminated PEG. Using optical absorbance spectroscopy and flow cytometry, we investigated the effect of PEG coating and molecular weight (MW) of PEG [5000 and 30,000 Daltons (Da)] on the phagocytic content of human spleen macrophages incubated with ICG-containing nanocapsules (ICG-NCs) between 15 to 360 min. Our results indicate that surface coating with PEG is an effective method to reduce the phagocytic content of ICG-NCs within macrophages for at least up to 360 min of incubation time. Coating the surface of ICG-NCs with the low MW PEG results in lower phagocytic content of ICG-NCs within macrophages for at least up to 60 min of incubation time as compared to ICG-NCs coated with the high MW PEG. Surface coating of ICG-NCs with PEG is a promising approach to prolong vasculature circulation time of ICG for NIR imaging and phototherapeutic applications.

Amyloid Histology Stain for Rapid Bacterial Endospore Imaging

Journal of Clinical Microbiology. Aug, 2011  |  Pubmed ID: 21653779

Bacterial endospores are some of the most resilient forms of life known to us, with their persistent survival capability resulting from a complex and effective structural organization. The outer membrane of endospores is surrounded by the densely packed endospore coat and exosporium, containing amyloid or amyloid-like proteins. In fact, it is the impenetrable composition of the endospore coat and the exosporium that makes staining methodologies for endospore detection complex and challenging. Therefore, a plausible strategy for facile and expedient staining would be to target components of the protective surface layers of the endospores. Instead of targeting endogenous markers encapsulated in the spores, here we demonstrated staining of these dormant life entities that targets the amyloid domains, i.e., the very surface components that make the coats of these species impenetrable. Using an amyloid staining dye, thioflavin T (ThT), we examined this strategy. A short incubation of bacillus endospore suspensions with ThT, under ambient conditions, resulted in (i) an enhancement of the fluorescence of ThT and (ii) the accumulation of ThT in the endospores, affording fluorescence images with excellent contrast ratios. Fluorescence images revealed that ThT tends to accumulate in the surface regions of the endospores. The observed fluorescence enhancement and dye accumulation, coupled with the sensitivity of emission techniques, provide an effective and rapid means of staining endospores without the inconvenience of pre- or posttreatment of samples.

Synthesis and Characterization of Bovine Serum Albumin-coated Nanocapsules Loaded with Indocyanine Green As Potential Multifunctional Nanoconstructs

Biotechnology Progress. Sep, 2011  |  Pubmed ID: 22002955

We have synthesized and characterized bovine serum albumin (BSA)-coated polymeric nanocapsules (NCs) loaded with indocyanine green (ICG), an FDA-approved near infrared chromophore. Poly(allylamine) hydrochloride was electrostatically crosslinked with phosphate anions to form nanoconstructs encapsulating ICG. BSA was conjugated onto the polymeric NCs via glutaraldehyde. BSA-coated ICG-containing nanocapsules (BSA-ICG NCs) were characterized by FTIR and optical spectroscopy, SEM, and zeta-potential measurements. Using normal human endocervical epithelial cells, we demonstrate the effectiveness of BSA-ICG NCs for intracellular optical imaging in vitro. These nanoconstructs may potentially serve as a multifunctional platform for combined optical imaging, phototherapy, and drug delivery. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011.

Biophotonics in Bioengineering

Annals of Biomedical Engineering. Jan, 2012  |  Pubmed ID: 22286899