We present a polarization-sensitive hyperspectral imaging system (SkinSpect) that employs a spectrally-programmable light source in the visible and NIR domains. Multiple tissue-mimicking phantoms were fabricated to mimic the optical properties of normal skin as well as pigmented light and dark moles. The phantoms consist of titanium dioxide and a mixture of coffee, red food dye, and naphthol green as the scattering and the three absorptive agents in a polydimethylsiloxane (PDMS) base. Phantoms were produced with both smooth and rough textured surfaces and tested using Spatial Frequency Domain Imaging (SFDI) and Spatially Modulated Quantitative Spectroscopy (SMoQS) for homogeneity as well as determining absorption and scattering variance, respectively. The reflectance spectral images were also recorded using the SkinSpect research prototype; the spectral signatures of the phantoms were calculated using a two-flux single-layer Kubelka-Munk model and non-negative least square fitting routine was applied to extract the relative concentrations of the individual phantom components. © 2013 Copyright SPIE.

A polarization-sensitive hyperspectral imaging system (SkinSpect) has been built and evaluated using two-layer tissue phantoms, fabricated to mimic the optical properties of melanin in different epidermal thickness and hemoglobin in dermal layers. Multiple tissue-mimicking phantoms with varying top layer thicknesses were measured for optical system calibration and performance testing. Phantom properties were characterized and validated using SkinSpect. The resulting analysis shows that the proposed system is capable of distinguishing and differentiating the layer-dependent absorption spectra and the depths at which this absorption occurs.

We have conducted time-resolved studies of optical breakdown produced by the irradiation water using 6 ns Nd:YAG laser pulses of 1064 nm and 532 nm wavelength focused at a numerical aperture of NA = 0.9. We determined pulse energy threshold values for plasma formation to be 1.89 μJ and 18.3 μJ for 532 and 1064 nm irradiation, respectively. These energy thresholds correspond to irradiance thresholds of 0.77 × 109 W/mm2 for 532 nm irradiation and 1.87 × 109 W/mm2 for 1064 nm irradiation. For pulse energies 1×, 2×, 5×, and 10× above threshold, we determined the length of the laser-induced plasma, the propagation speed and peak pressures of the emitted shock wave, and the mechanical energy dissipated by subsequent cavitation bubble formation, growth, and collapse. This analysis demonstrates that both the breakdown threshold as well as the conversion efficiency of the incident laser energy into mechanical energy is smaller for irradiation at 532 nm than for 1064 nm. These results are consistent with laser parameters employed for a variety of nanosecond pulsed microirradiation procedures using 1064 nm and 532 nm radiation focused by microscope objectives with large numerical apertures (NA ≳ 0.8). These results suggest that laser-induced breakdown is the primary mechanism that drives a variety of cellular micromanipulation techniques which employ nanosecond visible and near-infrared laser pulses.

We have developed a multimode dermoscope (SkinSpect™) capable of illuminating human skin samples in-vivo with spectrally-programmable linearly-polarized light at 33 wavelengths between 468nm and 857 nm. Diffusely reflected photons are separated into collinear and cross-polarized image paths and images captured for each illumination wavelength. In vivo human skin nevi (N = 20) were evaluated with the multimode dermoscope and melanin and hemoglobin concentrations were compared with Spatially Modulated Quantitative Spectroscopy (SMoQS) measurements. Both systems show low correlation between their melanin and hemoglobin concentrations, demonstrating the ability of the SkinSpectTM to separate these molecular signatures and thus act as a biologically plausible device capable of early onset melanoma detection.

In this study, we investigated the effects of size and surrounding media viscosity on trapping of microspheres. A continuous wave ytterbium fiber laser with a 1064 nm wavelength was used to create an optical tweezers system for optical manipulation experiments. Briefly, the system consisted of an inverted microscope, and a 100X 1.4 NA oil immersion objective through which the laser beam converged to form the optical trap. The laser beam was collimated, steered, and coupled to the microscope through the epifluorescence microscope port. The laser power at the trap focal spot was determined by measuring the input power at the back aperture of the objective multiplied by the objective transmission factor at 1064 nm measured by a modified dual objective method. Polystyrene microspheres varying in diameter from 5 to 15 microns were suspended in liquid media in glass bottom petri dishes prior to trapping experiments. The microspheres were trapped at different trapping powers, and fluidic viscous drag forces where applied to the optically trapped microspheres by driving a computer controlled 2D motorized microscope stage at known velocities. The drag forces were calculated at the point that the microspheres fell out of the trap, based on the Stokes equation for flow around spheres. The data show a linear relationship between trapping force and trap power within the range of the microsphere diameters and media viscosity values used. The work includes calculation of the dimensionless trap efficiency coefficient (Q) at 1064 nm wavelength and the corresponding effects of media viscosity and microsphere size on (Q). © 2014 SPIE.

Changes in the pattern and distribution of both melanocytes (pigment producing) and vasculature (hemoglobin containing) are important in distinguishing melanocytic proliferations. The ability to accurately measure melanin distribution at different depths and to distinguish it from hemoglobin is clearly important when assessing pigmented lesions (benign versus malignant). We have developed a multimode hyperspectral dermoscope (SkinSpect™) able to more accurately image both melanin and hemoglobin distribution in skin. SkinSpect uses both hyperspectral and polarization-sensitive measurements. SkinSpect’s higher accuracy has been obtained by correcting for the effect of melanin absorption on hemoglobin absorption in measurements of melanocytic nevi. In vivo human skin pigmented nevi (N=20) were evaluated with the SkinSpect, and measured melanin and hemoglobin concentrations were compared with spatial frequency domain spectroscopy (SFDS) measurements. We confirm that both systems show low correlation of hemoglobin concentrations with regions containing different melanin concentrations (R=0.13 for SFDS, R=0.07 for SkinSpect).

Lymphocytes are the central players in the human adaptive immune response. In the body, individual T helper lymphocytes need to be activated first by physical contact with antigen-presenting cells (APC). T-cell contact with APCs initiates an activation cascade, which includes an increase in T-cell intracellular calcium, leading to T-cell proliferation, differentiation and lymphokine production. Calcium imaging are combined with optical manipulation to investigate the physical properties of T-cell activation. We study cell-cell contact requirements for T-cell activation using optical tweezers to control the orientation of T-cell/APC pairs and fluorescence microscopy to measure the subsequent T-cell intracellular calcium level ([Ca2+]i) response. APCs or beads coated with antibodies to the T-cell receptor (TCR) are trapped with a near-infrared titanium-sapphire laser and placed at different locations along the T-cell, which has a polarized appearance defined by the shape and direction of crawling. T cells which are presented with antigen at the leading edge have a higher probability of responding and a shorter latency of response than those contacting APCs or beads with their trailing end. Alterations in antibody density and bead size are used to determine the spatial requirements for T cell activation and the minimum number of receptors which must be engaged in order to transmit a positive signal.

Highly focused laser microbeams are being used with increasing regularity for targeted cell lysis, cellular microsurgery and molecular delivery via transient cell membrane permeabilization. To examine the mechanisms of laser induced cell lysis, we performed time-resolved imaging of confluent PtK2 cell cultures following the delivery of a single 6 ns, 532 run Nd:YAG laser pulse. The laser pulse energies employed correspond to 1× and 3× threshold for plasma formation. The resulting plasma formation, pressure wave propagation and cavitation bubble dynamics were imaged over a temporal range spanning 5 orders of magnitude (0.5 ns - 50 μs). Time-resolved imaging enabled determination of process characteristics including pressure wave speed and amplitude and cavitation bubble energies. The time-resolved images also revealed the onset of cellular damage to occur on nano-second time scales and complete within 1 μs. Moreover, the size of the damage zone was larger than the plasma but smaller than the maximum cavitation bubble size. This indicated that mechanisms apart from plasma vaporization namely pressure wave propagation and cavitation bubble expansion are contributors to cellular damage. Dye exclusion assays showed that the majority of cells experiencing considerable deformation due to fluid flow generated by the cavitation bubble expansion remain viable over 24 hours.

Transport processes are important in biology and medicine. Examples include virus docking and infection, endocytosis of extracellular protein and phagocytosis of antigenic material. Trafficking driven by molecular motors inside a complex 3D environment is a shared common theme. The complex sequence of these events are difficult to resolve with conventional techniques where the action of many cells are asynchronously averaged. Single particle tracking (SPT) was developed by Ghosh and Webb to address this problem and has proven to be a powerful technique in understanding membrane- protein interaction. Since the traditional SPT method uses wide field illumination and area detectors, it is limited to the study of 2D systems. In this presentation, we report the development of a 3D single particle tracking technique using two-photon excitation. Using a real-time feedback system, we can dynamically position the sub-femtoliter two-photon excitation volume to follow the fluorescent particle under transport by maximizing the detected fluorescent intensity. Further, fluorescence spectroscopy can be performed in real time along the particle trajectory to monitor the underlying biochemical signals driving this transport process. The first application of this instrument will focus on the study of antigen endocytosis process of macrophages. ©2004 Copyright SPIE - The International Society for Optical Engineering.