Nicholas P. Barry

Fluorescence lifetime imaging for the two-photon microscope: time-domain and frequency-domain methods

Fluorescence lifetime images are obtained with the laser scanning microscope using two methods: the time-correlated single-photon counting method and the frequency-domain method. In the same microscope system, we implement both methods. We perform a comparison of the performance of the two approaches in terms of signal-to-noise ratio (SNR) and the speed of data acquisition. While in our practical implementation the time-correlated single-photon counting technique provides a better SNR for low-intensity images, the frequency-domain method is faster and provides less distortion for bright samples.

Altered renal tubular expression of the complement inhibitor Crry permits complement activation after ischemia/reperfusion

Ischemia/reperfusion (I/R) of several organs results in complement activation, but the kidney is unique in that activation after I/R occurs only via the alternative pathway. We hypothesized that selective activation of this pathway after renal I/R could occur either because of a loss of complement inhibition or from increased local synthesis of complement factors. We examined the relationship between renal complement activation after I/R and the levels and localization of intrinsic membrane complement inhibitors. We found that loss of polarity of complement receptor 1-related protein y (Crry) in the tubular epithelium preceded activation of the alternative pathway along the basolateral aspect of the tubular cells. Heterozygous gene-targeted mice that expressed lower amounts of Crry were more sensitive to ischemic injury. Furthermore, inhibition of Crry expressed by proximal tubular epithelial cells in vitro resulted in alternative pathway-mediated injury to the cells. Thus, altered expression of a complement inhibitor within the tubular epithelium appears to be a critical factor permitting activation of the alternative pathway of complement after I/R. Increased C3 mRNA and decreased factor H mRNA were also detected in the outer medulla after I/R, suggesting that altered synthesis of these factors might further contribute to complement activation in this location.

Detection of luminescent single ultrasmall silicon nanoparticles using fluctuation correlation spectroscopy

We dispersed electrochemical etched Si into a colloid of ultrasmall blue luminescent nanoparticles, observable with the naked eye, in room light. We use two-photon near-infrared femtosecond excitation at 780 nm to record the fluctuating time series of the luminescence, and determine the number density, brightness, and size of diffusing fluorescent particles. The luminescence efficiency of particles is high enough such that we are able to detect a single particle, in a focal volume, of 1 pcm3. The measurements yield a particle size of 1 nm, consistent with direct imaging by transmission electron microscopy. They also yield an excitation efficiency under two-photon excitation two to threefold larger than that of fluorescein. Detection of single particles paves the way for their use as labels in biosensing applications.

Stimulated blue emission in reconstituted films of ultrasmall silicon nanoparticles

We dispersed electrochemical etched Si into a colloid of ultrabright blue luminescent nanoparticles (1 nm in diameter) and reconstituted it into films or microcrystallites. When the film is excited by a near-infrared two-photon process at 780 nm, the emission exhibits a sharp threshold near 106 W/cm2, rising by many orders of magnitude, beyond which a low power dependence sets in. Under some conditions, spontaneous recrystallization forms crystals of smooth shape from which we observe collimated beam emission, pointing to very large gain coefficients. The results are discussed in terms of population inversion, produced by quantum tunneling or/and thermal activation, and stimulated emission in the quantum confinement-engineered Si–Si phase found only on ultrasmall Si nanoparticles. The Si–Si phase model provides gain coefficients as large as 103–105 cm−1.

Regulation of rat intestinal Na-dependent phosphate transporters by dietary phosphate

Hyperphosphatemia associated with chronic kidney disease is one of the factors that can promote vascular calcification, and intestinal P(i) absorption is one of the pharmacological targets that prevents it. The type II Na-P(i) cotransporter NaPi-2b is the major transporter that mediates P(i) reabsorption in the intestine. The potential role and regulation of other Na-P(i) transporters remain unknown. We have identified expression of the type III Na-P(i) cotransporter PiT-1 in the apical membrane of enterocytes. Na-P(i) transport activity and NaPi-2b and PiT-1 proteins are mostly expressed in the duodenum and jejunum of rat small intestine; their expression is negligible in the ileum. In response to a chronic low-P(i) diet, there is an adaptive response restricted to the jejunum, with increased brush border membrane (BBM) Na-P(i) transport activity and NaPi-2b, but not PiT-1, protein and mRNA abundance. However, in rats acutely switched from a low- to a high-P(i) diet, there is an increase in BBM Na-P(i) transport activity in the duodenum that is associated with an increase in BBM NaPi-2b protein abundance. Acute adaptive upregulation is restricted to the duodenum and induces an increase in serum P(i) that produces a transient postprandial hyperphosphatemia. Our study, therefore, indicates that Na-P(i) transport activity and NaPi-2b protein expression are differentially regulated in the duodenum vs. the jejunum and that postprandial upregulation of NaPi-2b could be a potential target for treatment of hyperphosphatemia.

Observation of laser oscillation in aggregates of ultrasmall silicon nanoparticles

We report laser oscillation at ∼610 nm in aggregates of ultrasmall elemental Si nanoparticles. The particles are ultrabright red emitting, dispersed from bulk Si by electrochemistry. The aggregates are excited by radiation at 550–570 nm from a mercury lamp. Intense directed Gaussian beams, with a threshold, manifest the emission. We observe line narrowing, and speckle patterns, indicating spatial coherence. This microlasing constitutes an important step towards the realization of a laser on a chip, hence optoelectronics integration and optical interconnects.

Multimodal CARS microscopy determination of the impact of diet on macrophage infiltration and lipid accumulation on plaque formation in ApoE-deficient mice

We characterized several cellular and structural features of early stage Type II/III atherosclerotic plaques in an established model of atherosclerosis—the ApoE-deficient mouse—by using a multimodal, coregistered imaging system that integrates three nonlinear optical microscopy (NLOM) contrast mechanisms: coherent anti-Stokes Raman scattering (CARS), second harmonic generation (SHG), and two-photon excitation fluorescence (TPEF). Specifically, the infiltration of lipid-rich macrophages and the structural organization of collagen and elastin fibers were visualized by CARS, SHG, and TPEF, respectively, in thick tissue specimens without the use of exogenous labels or dyes. Label-free CARS imaging of macrophage accumulation was confirmed by histopathology using CD68 staining. A high-fat, high-cholesterol Western diet resulted in an approximate 2-fold increase in intimal plaque area, defined by CARS signals of lipid-rich macrophages. Additionally, analysis of collagen distribution within lipid-rich plaque regions revealed nearly a 4-fold decrease in the Western diet–fed mice, suggesting NLOM sensitivity to increased matrix metalloproteinase (MMP) activity and decreased smooth muscle cell (SMC) accumulation. These imaging results provide significant insight into the structure and composition of early stage Type II/III plaque during formation and allow for quantitative measurements of the impact of diet and other factors on critical plaque and arterial wall features.

Mitigating thermal mechanical damage potential during two-photon dermal imaging

Two-photon excitation fluorescence microscopy allows in vivo high-resolution imaging of human skin structure and biochemistry with a penetration depth over 100 microm. The major damage mechanism during two-photon skin imaging is associated with the formation of cavitation at the epidermal-dermal junction, which results in thermal mechanical damage of the tissue. In this report, we verify that this damage mechanism is of thermal origin and is associated with one-photon absorption of infrared excitation light by melanin granules present in the epidermal-dermal junction. The thermal mechanical damage threshold for selected Caucasian skin specimens from a skin bank as a function of laser pulse energy and repetition rate has been determined. The experimentally established thermal mechanical damage threshold is consistent with a simple heat diffusion model for skin under femtosecond pulse laser illumination. Minimizing thermal mechanical damage is vital for the potential use of two-photon imaging in noninvasive optical biopsy of human skin in vivo. We describe a technique to mitigate specimen thermal mechanical damage based on the use of a laser pulse picker that reduces the laser repetition rate by selecting a fraction of pulses from a laser pulse train. Since the laser pulse picker decreases laser average power while maintaining laser pulse peak power, thermal mechanical damage can be minimized while two-photon fluorescence excitation efficiency is maximized.

Highly nonlinear photoluminescence threshold in porous silicon

Porous silicon is excited using near-infrared femtosecond pulsed and continuous wave radiation at an average intensity of ∼106 W/cm2 (8×1010 W/cm2 peak intensity in pulsed mode). Our results demonstrate the presence of micron-size regions for which the intensity of the photoluminescence has a highly nonlinear threshold, rising by several orders of magnitude near this incident intensity for both the pulsed and continuous wave cases. These results are discussed in terms of stimulated emission from quantum confinement engineered intrinsic Si–Si radiative traps in ultrasmall nanocrystallites, populated following two-photon absorption.

Si–N linkage in ultrabright, ultrasmall Si nanoparticles

Ultrabright ultrasmall (∼1 nm) blue luminescent Si29 nanoparticles are chlorinated by reaction with Cl2 gas. A Si–N linkage is formed by the reaction of the chlorinated particles with the functional amine group in butylamine. Fourier transform infrared spectroscopy and x-ray photospectroscopy measurements confirm the N linkage and the presence of the butyl group, while emission, excitation, and autocorrelation femtosecond optical spectroscopy show that, after the linkage formation, the particles with the ultrabright blue luminescent remain, but with a redshift of 40 nm.