K.-C. Chiu

Enhanced live cell membrane imaging using surface plasmon-enhanced total internal reflection fluorescence microscopy.

Using a total internal reflection fluorescence microscopy (TIRFM) technique to image live cells on a biosurface not only provides an enhanced understanding of cellular functions, but also improves the signal-to-noise ratio of the images. However, the intensity of the fluorescence signal must be increased if a more dynamic biomolecular imaging capability is required. Accordingly, this study presents a surface plasmon-enhanced TIRFM technique in which the fluorescence signals are enhanced via surface plasmons offered by a silver nanolayer. The developed microscopy technique is successfully applied to the real-time observation of the thrombomodulin proteins of live cell membranes. The experimental results and the simulation results demonstrate that the live cell membrane images obtained in the proposed surface plasmon-enhanced TIRFM technique are brighter by approximately one order of magnitude than those provided by conventional TIRFM.

Optimizing silver film for surface plasmon-coupled emission induced two-photon excited fluorescence imaging

In this study, the optimal condition of a silver (Ag) film deposited on a cover slip for surface plasmon-coupled emission (SPCE) induced two-photon excited fluorescence (TPEF) based on an objective-based, total internal reflection (TIR) microscope was investigated. According to the theoretical simulations of local electric field enhancement and fluorescence coupled emission efficiency, the thickness of the Ag film should be about 40 nm in order to maximize the TPEF collection efficiency by the objective. The deposited Ag film with a germanium seed layer on a cover slip exhibits additional improvement in surface smoothness by reducing variations in surface roughness to below 1.0 nm, thereby reduces local hot spots which degrade the image uniformity. Moreover, an Ag film with a 20 nm-thick SiO2 spacer not only prevents damage caused through interaction with the aqueous solution under high laser power irradiance, but also reduces the fluorescence quenching effect by the Ag film. By optimizing the Ag film thickness, surface smoothness, and a protective dielectric spacer, efficient TIR TPEF imaging can be achieved through SPCE.

Surface plasmon resonance biosensors with subwavelength grating waveguide

In this study, a surface plasmon resonance (SPR) biosensor with sub-wavelength grating waveguide for the real-time analysis of biomolecular interactions is developed. The conventional SPR has diffractive grating structure to increase the wave vector for exciting the surface plasmons and then detects biomolecular interactions in high order diffraction light. Using this approach has some disadvantages such as the intensity of high order diffraction light is dimmer to be difficult to measure and the measured reflectivity spectrum is too broadened. The proposed SPR biosensor uses a normally incident white light with the help of subwavelength grating structure and provides a sharper reflectivity spectrum according to waveguide interference both to avoid disadvantages of the conventional SPR biosensor with a grating coupler. When the diffraction grating waveguide structure and the condition of SPR are destroyed by external factors such as slight refractive index changes of the buffer or molecule adsorption on the grating surface, the optical path and momentum of the light coupled through the gold grating into the waveguide are changed and a resonance wavelength shift is induced as a result. By detecting this resonance wavelength shift, the SPR biosensor provides the ability to identify the kinetics of the biomolecular interaction on an on-line basis without the need for the extrinsic labeling of the biomolecules. The proposed biosensing metrology system becomes more simply and convenient for real-time biomolecular interaction analysis.

An investigation into the influence of secondary structures for DNA hybridization using surface plasmon resonance and surface-enhanced Raman scattering

This study utilizes a surface plasmon resonance (SPR) biosensing to investigate the influence of secondary structures on the DNA hybridization and a surface-enhanced Raman scattering (SERS) spectrum to yield analytical data regarding the structure of the oligonucleotides. It is found that the SPR angular shifts associated with the three pairs of 60mer oligonucleotides with prominent secondary structures are lower than those observed for the two pairs of oligonucleotides with no obvious secondary structures. It is also determined that increasing the DNA hybridization temperature from 35 oC to 45 oC reduces secondary structure effects. On the hybridization with mixture target oligonucleotides, the SPR results demonstrate that secondary structures interfere significantly. Although the kinetics of biomolecular interaction analysis is performed by using SPR sensor, the structural information of the oligonucleotides can not observed directly. The SERS spectrum provides the structural information of the oligonucleotides with silver colloidal nanoparticles adapted as a Raman active substrate. Also, the detection limit of the DNA Raman signal has been successfully improved to reach sub-micro molarity of DNA concentration.

Surface plasmon-enhanced and quenched two-photon excited fluorescence

This study investigated theoretically and experimentally that two-photon excited fluorescence is enhanced and quenched via surface plasmons (SPs) excited by total internal reflection with a silver film. The fluorescence intensity is fundamentally affected by the local electromagnetic field enhancement and the quantum yield change according to the surrounding structure and materials. By utilizing the Fresnel equation and classical dipole radiation modeling, local electric field enhancement, fluorescence quantum yield, and fluorescence emission coupling yield via SPs were theoretically analyzed at different dielectric spacer thicknesses between the fluorescence dye and the metal film. The fluorescence lifetime was also decreased substantially via the quenching effect. A two-photon excited total internal reflection fluorescence (TIRF) microscopy with a time-correlated single photon counting device has been developed to measure the fluorescence lifetimes, photostabilities, and enhancements. The experimental results demonstrate that the fluorescence lifetimes and the trend of the enhancements are consistent with the theoretical analysis. The maximum fluorescence enhancement factor in the surface plasmon-total internal reflection fluorescence (SP-TIRF) configuration can be increased up to 30 fold with a suitable thickness SiO2 spacer. Also, to compromise for the fluorescence enhancement and the fluorophore photostability, we find that the SP-TIRF configuration with a 10 nm SiO2 spacer can provide an enhanced and less photobleached fluorescent signal via the assistance of enhanced local electromagnetic field and quenched fluorescence lifetime, respectively.

Investigating the structural changes of β-amyloid peptide aggregation using attenuated-total-reflection surface-enhanced raman spectroscopy

This study utilizes a surface-enhanced Raman spectroscopy (SERS) based on the attenuated-total-reflection (ATR) method to investigate that the structural information of the biomolecular monolayer on sensing surface can be dynamically observed with a higher signal-to-noise ratio signal. The secondary structures of long oligonucleotides and their influence on the DNA hybridization on the sensing surface are investigated. The SERS spectrum provides the structural information of the oligonucleotides with the help of a silver colloidal nanoparticle monolayer by control of the size and distribution of the nanoparticles adapted as a Raman active substrate. It is found that the ring-breathing modes of adenine, thymine, guanine, and cytosine in Raman fingerprint associated with three 60mer oligonucleotides with prominent secondary structures are lower than those observed for the two oligonucleotides with no obvious secondary structures. It is also determined that increasing the DNA hybridization temperature from 35°C to 45°C reduces secondary structure effects. The ATR-SERS biosensing technique will be used to provide valuable structural information regarding the short-term reversible interactions and long-term polymerization events in the A&bgr; aggregates on the sensing surface.

Investigating the secondary structures for long oligonucleotides using attenuated-total-reflection nanoplasmon-enhanced Raman scattering

This study utilizes a nanoplasmon-enhanced Raman scattering based on the attenuated-total-reflection (ATR) method to investigate the secondary structures of long oligonucleotides and their influence on the DNA hybridization. It is found that the ring-breathing modes of adenine, thymine, guanine, and cytosine in Raman fingerprint associated with three 60mer oligonucleotides with prominent secondary structures are lower than those observed for the two oligonucleotides with no obvious secondary structures. It is also determined that increasing the DNA hybridization temperature from 35 oC to 45 oC reduces secondary structure effects. The kinetics of biomolecular interaction analysis can be performed by using surface plasmons resonance biosensor, but the structural information of the oligonucleotides can not observed directly. The ATR-Raman spectrum can provide the structural information of the oligonucleotide monolayer on the sensing surface with the help of a silver patterned nanostructure film based on the finite-difference time-domain simulation and the e-beam lithography fabrication adapted as an ATR-Raman active substrate.

STUDY OF NANOPRISMS VIA APERTURELESS NEAR-FIELD OPTICAL MICROSCOPY

To study nano-scale optical local-field phenomena, an apertureless near-field scanning optical microscope (aNSOM) is an important tool. Herein, an aNSOM has been developed and is utilized for observing the local surface plasmon resonance, wave propagation, and nano-antenna enhancement of nanoprisms. The developed aNSOM, based on a commercial atomic force microscope, is integrated with homodyne and heterodyne interferometric techniques to detect the near-field amplitude and phase of nanostructures. With the help of mechanical system designs, different illumination direction s and detections for different applications can be achieved.

2D freeform plasmonic trapping via spatial light modulator

In this study, an objective-based two-dimensional (2D) surface plasmon (SP)-enhanced optical trapping system with a spatial light modulator (SLM) has been developed to trap dielectric particles in freeform pattern. Through a gold film with a thickness of 45 nm in the near infrared region, a 40-fold electric field enhancement is reached and hence a strong 2D trapping force distribution with SP excitation has been demonstrated. Furthermore, the algorithm called weighted Gerchberg-Saxton can provide a freeform pattern which is used to control the trapping force distributions in the image space based on SLM. Unlike the patterns formed by finite gold areas fabricated on a glass surface, the freeform plasmonic trapping is a more convenient and efficient method to manipulate nanoparticles and biomolecules arbitrarily.

Surface plasmonic microscopy for live cell membrane imaging

This study presents a surface plasmon-enhanced total internal reflection fluorescence microscopy (TIRFM) and a surface plasmon polariton (SPP) phase microscopy techniques to image live cell membranes. In the surface plasmon-enhanced TIRFM, the developed microscopy technique is successfully applied to the real-time observation of the thrombomodulin proteins of live cell membranes.