Jing Yong Ye

Adaptive correction of depth-induced aberrations in multiphoton scanning microscopy using a deformable mirror

We demonstrate adaptive aberration correction for depth‐induced spherical aberration in a multiphoton scanning microscope with a micromachined deformable mirror. Correction was made using a genetic learning algorithm with two‐photon fluorescence intensity feedback to determine the desired shape for an adaptive mirror. For a 40×/0.6 NA long working distance objective, the axial scanning range was increased from 150 mm to 600 mm.

Enhanced two-photon biosensing with double-clad photonic crystal fibers

A double-clad photonic crystal fiber was used to improve detection efficiency over a standard single-mode fiber in a two-photon fluorescence detection scheme in which the dye was excited and the fluorescence was detected back through the same fiber.

Controlled permeation of cell membrane by single bubble acoustic cavitation

Sonoporation is the membrane disruption generated by ultrasound and has been exploited as a non-viral strategy for drug and gene delivery. Acoustic cavitation of microbubbles has been recognized to play an important role in sonoporation. However, due to the lack of adequate techniques for precise control of cavitation activities and real-time assessment of the resulting sub-micron process of sonoporation, limited knowledge has been available regarding the detail processes and correlation of cavitation with membrane disruption at the single cell level. In the current study, we developed a combined approach including optical, acoustical, and electrophysiological techniques to enable synchronized manipulation, imaging, and measurement of cavitation of single bubbles and the resulting cell membrane disruption in real-time. Using a self-focused femtosecond laser and high frequency ultrasound (7.44MHz) pulses, a single microbubble was generated and positioned at a desired distance from the membrane of a Xenopus oocyte. Cavitation of the bubble was achieved by applying a low frequency (1.5MHz) ultrasound pulse (duration 13.3 or 40μs) to induce bubble collapse. Disruption of the cell membrane was assessed by the increase in the transmembrane current (TMC) of the cell under voltage clamp. Simultaneous high-speed bright field imaging of cavitation and measurements of the TMC were obtained to correlate the ultrasound-generated bubble activities with the cell membrane poration. The change in membrane permeability was directly associated with the formation of a sub-micrometer pore from a local membrane rupture generated by bubble collapse or bubble compression depending on ultrasound amplitude and duration. The impact of the bubble collapse on membrane permeation decreased rapidly with increasing distance (D) between the bubble (diameter d) and the cell membrane. The effective range of cavitation impact on membrane poration was determined to be D/d=0.75. The maximum mean radius of the pores was estimated from the measured TMC to be 0.106±0.032μm (n=70) for acoustic pressure of 1.5MPa (duration 13.3μs), and increased to 0.171±0.030μm (n=125) for acoustic pressure of 1.7MPa and to 0.182±0.052μm (n=112) for a pulse duration of 40μs (1.5MPa). These results from controlled cell membrane permeation by cavitation of single bubbles revealed insights and key factors affecting sonoporation at the single cell level.

Real-time biomolecular binding detection using a sensitive photonic crystal biosensor.

Real-time measurement of specific biomolecular interactions is critical to many areas of biological research. A number of label-free techniques for directly monitoring biomolecular binding have been developed, but it is still challenging to measure the binding kinetics of very small molecules, to detect low concentrations of analyte molecules, or to detect low affinity interactions. In this study, we report the development of a highly sensitive photonic crystal biosensor for label-free, real-time biomolecular binding analysis. We characterize the performance of this biosensor using a standard streptavidin-biotin binding system. Optimization of the surface functionalization methods for streptavidin immobilization on the silica sensing surface is presented, and the specific binding of biotinylated analyte molecules ranging over 3 orders of magnitude in molecular weight, including very small molecules (<250 Da), DNA oligonucleotides, proteins, and antibodies (>150 000 Da), are detected in real time with a high signal-to-noise ratio. Finally, we document the sensor efficiency for low mass adsorption, as well as multilayered molecular interactions. By all important metrics for sensitivity, we anticipate this photonic crystal biosensor will provide new capabilities for highly sensitive measurements of biomolecular binding.

Sensitive molecular binding assay using a photonic crystal structure in total internal reflection

A novel optical sensor for label-free biomolecular binding assay using a one-dimensional photonic crystal in a total-internal-reflection geometry is proposed and demonstrated. The simple configuration provides a narrow optical resonance to enable sensitive measurements of molecular binding, and at the same time employs an open interface to enable real-time measurements of binding dynamics. Ultrathin aminopropyltriethoxysilane/ glutaraldehyde films adsorbed on the interface were detected by measuring the spectral shift of the photonic crystal resonance and the intensity ratio change in a differential reflectance measurement. A detection limit of 6 x 10(-5) nm for molecular layer thickness was obtained, which corresponds to a detection limit for analyte adsorption of 0.06 pg/mm(2) or a refractive index resolution of 3 x 10(-8) RIU; this represents a significant improvement relative to state-of-the-art surface-plasmon-resonance-based systems.

Enhancement of two-photon excited fluorescence using one-dimensional photonic crystals

We fabricated a one-dimensional photonic crystal structure with a defect layer of a poly(vinyl alcohol) thin film doped with 2-aminopurine (2AP). The defect induced a transmission peak in the photonic band gap at 610 nm, to which ultrashort laser pulses were tuned. We observed enhanced two-photon fluorescence emission from 2AP in the photonic crystal structure with a factor of 120. The enhancement was attributed to the high local field of light generated by a photonic state localized at the defect layer. Furthermore, under the enhanced light intensity, we carried out photobleaching experiments, which gave useful information on the photochemistry of 2AP.

Association of Acinetobacter baumannii EF-Tu with cell surface, outer membrane vesicles, and fibronectin

A conundrum has long lingered over association of cytosol elongation factor Tu (EF-Tu) with bacterial surface. Here we investigated it with Acinetobacter baumannii, an emerging opportunistic pathogen associated with a wide spectrum of infectious diseases. The gene for A. baumannii EF-Tu was sequenced, and recombinant EF-Tu was purified for antibody development. EF-Tu on the bacterial surface and the outer membrane vesicles (OMVs) was revealed by immune electron microscopy, and its presence in the outer membrane (OM) and the OMV subproteomes was verified by Western blotting with the EF-Tu antibodies and confirmed by proteomic analyses. EF-Tu in the OM and the OMV subproteomes bound to fibronectin as detected by Western blot and confirmed by a label-free real-time optical sensor. The sensor that originates from photonic crystal structure in a total-Internal-reflection (PC-TIR) configuration was functionalized with fibronectin for characterizing EF-Tu binding. Altogether, with a novel combination of immunological, proteomical, and biophysical assays, these results suggest association of A. baumannii EF-Tu with the bacterial cell surface, OMVs, and fibronectin.

Biosensing based on two-photon fluorescence measurements through optical fibers.

We have performed two-photon fluorescence detection in a new scheme in which femtosecond laser pulses were delivered thorugh an optical fiber for nonlinear excitation and the emitted fluorescence was collected through the same fiber. Single-mode fibers were determined to give higher detection efficiency than multimode fibers, consistent with theoretical considerations. The utility of fiber-optic sensing based on two-photon fluorescence detection was proved by an experiment that measured the uptake of a targeted drug delivery agent into cultured cancer cells.

Single-molecule imaging and spectroscopy of adenine and an analog of adenine using surface-enhanced Raman scattering and fluorescence

Abstract We implemented Raman imaging and spectroscopy of adenine molecules adsorbed on Ag colloidal nanoparticles to find feasibility of detecting and identifying nucleic-acid bases at the single-molecule level. Surface enhanced Raman scattering of adenine molecules showed an intermittent on-and-off behavior called “blinking” and fluctuation of the center of spectra called “spectral diffusion”. The observation of blinking and spectral diffusion provides substantial evidence for detecting single adenine molecules. Also, we extended single molecule fluorescence imaging and time-resolved fluorometry from green to violet-excitation regime to detect a fluorescent analog of nucleic-acid bases at the single-molecule level. Using violet excitation we observed fluorescence spectra and lifetimes from single complexes of an analog of adenine and the Klenow fragment of DNA polymerase I.

Enhancement of laser-induced optical breakdown using metal/dendrimer nanocomposites

We demonstrate that dendrimer nanocomposites (DNC) can be used to remarkably change the laser-induced optical breakdown (LIOB) threshold of a material, owing to a large enhancement of the local electric field. We have implemented LIOB using femtosecond laser pulses in a gold/dendrimer hybrid nanocomposite as a model system. Third-harmonic generation measurements have been employed as a sensitive way for monitoring the LIOB in situ and in real time. The observed statistical behavior of the breakdown process is attributed to a laser-driven aggregation of individual DNC particles. The breakdown threshold value of the DNC has been found to be up to two orders of magnitude lower than that of pure dendrimers or normal tissues.