Arnold D. Estrada

Two-photon-induced photoluminescence imaging of tumors using near-infrared excited gold nanoshells

We report strong two-photon-induced photoluminescence (TPIP) from silica/gold nanoshells (NS). We demonstrate its potential application for imaging the 3D distribution of NS in tumors using a NIR laser scanning multi-photon microscope.

Intra-organ Biodistribution of Gold Nanoparticles Using Intrinsic Two-photon Induced Photoluminescence

Gold nanoparticles (GNPs) such as gold nanoshells (GNSs) and gold nanorods (GNRs) have been explored in a number of in vitro and in vivo studies as imaging contrast and cancer therapy agents due to their highly desirable spectral and molecular properties. While the organ‐level biodistribution of these particles has been reported previously, little is known about the cellular level or intra‐organ biodistribution. The objective of this study was to demonstrate the use of intrinsic two‐photon‐induced photoluminescence (TPIP) to study the cellular level biodistribution of GNPs.

Microvascular oxygen quantification using two-photon microscopy

An instrument is demonstrated that is capable of three-dimensional (3D) vasculature imaging and pO(2) quantification with high spatial resolution. The instrument combines two-photon (2P) microscopy with phosphorescence quenching to measure pO(2). The instrument was demonstrated by performing depth-resolved microvascular pO(2) measurements of rat cortical vessels down to 120 microm below the surface. 2P excitation of porphyrin was confirmed, and measured pO(2) values were consistent with previously published data for normoxic and hyperoxic conditions. The ability to perform 3D pO(2) measurements using optical techniques will allow researchers to overcome existing limitations imposed by polarographic electrodes, magnetic resonance techniques, and surface-only pO(2) measurement techniques.

Depth-resolved blood oxygen saturation measurement by dual-wavelength photothermal (DWP) optical coherence tomography

Non-invasive depth-resolved measurement of hemoglobin oxygen saturation (SaO2) levels in discrete blood vessels may have implications for diagnosis and treatment of various pathologies. We introduce a novel Dual-Wavelength Photothermal (DWP) Optical Coherence Tomography (OCT) for non-invasive depth-resolved measurement of SaO2 levels in a blood vessel phantom. DWP OCT SaO2 is linearly correlated with blood-gas SaO2 measurements. We demonstrate 6.3% precision in SaO2 levels measured a phantom blood vessel using DWP-OCT with 800 and 765 nm excitation wavelengths. Sources of uncertainty in SaO2 levels measured with DWP-OCT are identified and characterized.

Three-dimensional mapping of oxygen tension in cortical arterioles before and after occlusion.

Occlusions in single cortical microvessels lead to a reduction in oxygen supply, but this decrement has not been able to be quantified in three dimensions at the level of individual vessels using a single instrument. We demonstrate a combined optical system using two-photon phosphorescence lifetime and fluorescence microscopy (2PLM) to characterize the partial pressure of oxygen (pO2) in single descending cortical arterioles in the mouse brain before and after generating a targeted photothrombotic occlusion. Integrated real-time Laser Speckle Contrast Imaging (LSCI) provides wide-field perfusion maps that are used to monitor and guide the occlusion process while 2PLM maps changes in intravascular oxygen tension. We present the technique’s utility in highlighting the effects of vascular networking on the residual intravascular oxygen tensions measured after occlusion in three dimensions.

Improved sensitivity for two-photon frequency-domain lifetime measurement

We demonstrate a method to improve the measurement sensitivity of two-photon frequency-domain lifetime measurements in poor signal to background conditions. This technique uses sinusoidal modulation of the two-photon excitation source and detection of the second harmonic of the modulation frequency that appears in the emission. Additionally, we present the mathematical model which describes how the observed phase shift and amplitude demodulation factor of two-photon phosphorescence emission are related to the phosphorescence lifetime and modulation frequency. We demonstrate the validity of the model by showing the existence of new frequency terms in the phosphorescence emission generated from the quadratic nature of two-photon absorption and by showing that the phase shift and demodulation match theory for all frequency components.

In situ process monitoring in selective laser sintering using optical coherence tomography

Abstract. Selective laser sintering (SLS) is an efficient process in additive manufacturing that enables rapid part production from computer-based designs. However, SLS is limited by its notable lack of in situ process monitoring when compared with other manufacturing processes. We report the incorporation of optical coherence tomography (OCT) into an SLS system in detail and demonstrate access to surface and subsurface features. Video frame rate cross-sectional imaging reveals areas of sintering uniformity and areas of excessive heat error with high temporal resolution. We propose a set of image processing techniques for SLS process monitoring with OCT and report the limitations and obstacles for further OCT integration with SLS systems.

Optical coherence tomography image-guided smart laser knife for surgery

Surgical oncology can benefit from specialized tools that enhance imaging and enable precise cutting and removal of tissue without damage to adjacent structures. The combination of high‐resolution, fast optical coherence tomography (OCT) co‐aligned with a nanosecond pulsed thulium (Tm) laser offers advantages over conventional surgical laser systems. Tm lasers provide superior beam quality, high volumetric tissue removal rates with minimal residual thermal footprint in tissue, enabling a reduction in unwanted damage to delicate adjacent sub‐surface structures such as nerves or micro‐vessels. We investigated such a combined Tm/OCT system with co‐aligned imaging and cutting beams—a configuration we call a “smart laser knife.”

1940 nm all-fiber Q-switched fiber laser

We present development of a nanosecond Q-switched Tm3+-doped fiber laser with 16 W average power and 4.4 kW peak power operating at 1940 nm. The laser has a master oscillator power amplifier design, and uses large mode area Tm3+-doped fibers as the gain medium. Special techniques are used to splice Tm3+-doped fibers to minimize splice loss. The laser design is optimized to reduce non-linear effects, including modulation instability. Pulse width broadening due to high gain is observed and studied in detail. Medical surgery is a field of application where this laser may be able to improve clinical practice. The laser together with scanning galvanometer mirrors is used to cut precisely around small footprint vessels in tissue phantoms without leaving any visible residual thermal damage. These experiments provide proof-of-principle that this laser has promising potential in the laser surgery application space.

Two-Photon-Induced Photoluminescence Imaging of Gold Nanoshell's Tumor Biodistribution

Given their tunable optical properties and high optical absorption and scattering cross sections, gold nanoshells (GNS) have been explored for a number of in vitro and in vivo imaging contrast and cancer therapy agents. While it has been shown that GNSs preferentially accumulate at the tumor site, little is known about the accumulation kinetics within the tumor. We demonstrate accumulation kinetics of GNSs in bulk tumors and histology slides using two-photon induced photoluminescence (TPIP) imaging. We found that GNSs had a heterogeneous distribution with higher accumulation at the tumor cortex. In addition, GNSs were observed in unique patterns surrounding the perivascular region. These results demonstrate that direct luminescence based imaging of metal nanoparticles provides high resolution and molecular specific multiplexed images.