Y.-D. Su

Common-path phase-shift interferometry surface plasmon resonance imaging system

Surface plasmon resonance (SPR) and common-path phase-shift interferometry (PSI) techniques are integrated in a biosensing imaging system for measuring the two-dimensional spatial phase variation caused by biomolecular interactions on a sensing chip without the need for additional labeling. The common-path PSI technique has the advantage of long-term stability, even when it is subjected to external disturbances. Hence the system meets the requirements of the real-time kinetic studies involved in biomolecular interaction analysis. The proposed SPR-PSI imaging system demonstrates a detection limit of a 2 x 10(-7) refractive-index change, a long-term phase stability of 2.5 x 10(-4) pi rms for 4 h, and a spatial phase resolution of 10(-3) pi with a lateral resolution of 100 microm.

Study of cell-biosubstrate contacts via surface plasmon polariton phase microscopy

This study utilized a developed surface plasmon polariton (SPP) phase microscopy to observe cell-biosubstrate contacts. The developed SPP phase microscopy is highly sensitive to cell membrane contact with biosubstrates and also provides long-term phase stability to achieve time-lapse living cell observation. As such, an SPP intensity and phase sensitivity comparison demonstrates that the sensitivity of the phase measurement can be 100-fold greater than that of the intensity measurement. Also, a more than 2-hour cell apoptosis observation via the SPP phase microscopy is presented. To implement the incident angle from 70° to 78°, cell-biosubstrate contact images corresponding to the surface plasmon resonance (SPR) angles are obtained by utilizing the SPP phase measurement. According to the information of the corresponding SPR angle image and a multilayer simulation, the contact distances between a living melanoma cell and a bovine serum albumin substrate at four different locations have been estimated.

Fabrication of gold nanorods-doped, bovine serum albumin microstructures via multiphoton excited photochemistry

In this study, three-dimensional (3D) crosslinked bovine serum albumin (BSA) microstructures containing gold nanorods (AuNRs) were fabricated via multiphoton excited photochemistry using Rose Bengal (RB) as the photoactivator. To retain AuNRs in the 3D crosslinked BSA microstructures, the laser wavelength was chosen for two-photon RB absorption for improved two-photon crosslinking efficiency, but not for enhancing the longitudinal plasmon resonance of AuNRs which may result in photothermal damage of AuNRs. Furthermore, with two-photon excitation of RB via AuNRs plasmonics, the laser power can be reduced by about 30%. As a result, 3D BSA microstructures containing AuNRs can be successfully fabricated. The AuNRs-doped BSA microstructures can be applied in biomedical scaffolds with plasmonic properties such as two-photon luminescence imaging and photothermal therapy.

Imaging the cell membrane with surface plasmon resonance phase microscopy

Molecular interactions occurring on or near cell membrane surfaces are expected to have different properties from those occurring in bulk solutions. In order to analyze molecular interactions between the cell membrane with biomolecules having no additional fluorescence labeling, a microscope based on the integration of surface plasmon resonance (SPR) and common-path phase-shift interferometry (PSI) techniques is developed and used to study the cell adhesion and migration on the biosurfaces. The surface plasmons are excited by light via the attenuated total reflection method. The common-path PSI technique has features of long-term stability, even when subjected to external disturbances. Hence, the developed SPR phase microscope meets the requirements of real-time kinetic imaging. The proposed common-path SPR-PSI microscope demonstrates a detection limit of 2x10-7 refractive index unit and a long-term phase stability of 2.5x10-4 π root mean square over four hours. The developed microscope is successfully applied to the real-time observation the live cell membranes with thrombomodulin proteins.

Surface plasmon-enhanced two-photon fluorescence microscopy for live cell membrane imaging

A surface plasmon-enhanced two-photon total-internal-reflection fluorescence (TIRF) microscope has been developed to provide the fluorescent images of living cell membranes. The proposed microscope with the helps of surface plasmons (SPs) not only provides brighter fluorescent images based on the mechanism of local electromagnetic field enhancement, but also reduces photobleaching due to having shorter fluorophore lifetime. In comparison with one-photon TIRF, the two-photon TIRF can achieve higher signal-to-noise ratio cell membrane imaging due its smaller excitation volume and lower scattering. Combining with the SP enhancement and two-photon excitation TIRF, the microscope has demonstrated the brighter and more contrast fluorescence membrane images of living monkey kidney COS-7 fibroblasts transfected with an EYFP-MEM or EGFP-WOX1 construct.

A surface plasmon phase imaging system with subwavelength grating

A surface plasmon polariton (SPP) phase imaging microscope with a sub-wavelength grating structure is developed for high-resolution in-plane image measurement, which can be used on biological samples. Conventionally, most of SPP image systems use prism couplers to induce surface plasmons (SPs), but this approach has some drawbacks such as non-normal incident light producing optical aberration in imaging and making the metrology instrument more complicate. It can be improved by utilizing a normal incident light to excite the SPs through subwavelength grating structure, which replaces the prism so that it can observe in-plane sample on the sensing surface and simplify the instrument. Instead of measuring the intensity of the reflectivity, the phase measurement with higher sensitivity is proposed. In this study, the proposed SPP microscope integrates a common-path phase-shift interferometry (PSI) technique to obtain the two-dimensional spatial phase variation caused by biomolecular interactions on the sensing surface without requiring additional labeling. The common-path PSI technique provides long-term stability, even when it is subjected to external disturbances, to match the requirements of biomolecular interaction analysis. The system is presented as a high stability, high sensitivity, and in-plane SPP phase image.

A surface plasmon polariton phase microscope with a subwavelength grating structure

A surface plasmon polariton (SPP) phase imaging microscope with a subwavelength grating structure is developed for high-resolution in-plane image measurement, which can be used on biological samples. Conventionally, most of SPP image systems use prism couplers to induce surface plasmons (SPs), but this approach has some drawbacks such as non-normal incident light producing optical aberration in imaging and making the metrology instrument more complicate. It can be improved by utilizing a normal incident light to excite the SPs through subwavelength grating structure, which replaces the prism so that it can observe in-plane sample on the sensing surface and simplify the instrument. Instead of measuring the intensity of the reflectivity, the phase measurement with higher sensitivity is proposed. In this study, the proposed SPP microscope integrates a common-path phase-shift interferometry (PSI) technique to obtain the two-dimensional spatial phase variation caused by biomolecular interactions on the sensing surface without requiring additional labeling. The common-path PSI technique provides long-term stability, even when it is subjected to external disturbances, to match the requirements of biomolecular interaction analysis. The microscope is presented as a high stability, high sensitivity, and in-plane SPP phase image.

Widefield multiphoton excited fluorescence microscopy for animal study in vivo

Unlike conventional multiphoton excited microscopy according to pixel-by-pixel point scanning, a widefield multiphoton excited microscopy based on spatiotemporal focusing has been developed to construct three-dimensional (3D) multiphoton fluorescence images only with the need of an axial scanning. By implementing a 4.0 W 10 kHz femtosecond laser amplifier with an instant strong peak power and a fast TE-cooled EMCCD camera with an ultra-sensitive fluorescence detection, the multiphoton excited fluorescence images with the excitation area over 100 μm x 100 μm can be achieved at a frame rate up to 80 Hz. A mechanical shutter is utilized to control the exposure time of 1 ms, i.e. average ten laser pulses reach the fluorescent specimen, and hence an uniform enough multiphoton excited fluorescence image can be attained with less photobleaching. The Brownian motion of microbeads and 3D neuron cells of a rat cerebellum have been observed with a lateral spatial resolution of 0.24 μm and an axial resolution of 2.5 μm. Therefore, the developed widefield multiphoton microscopy can provide fast and high-resolution multiphoton excited fluorescence images for animal study in vivo.

A full-field heterodyne surface plasmon resonance dynamic bio-imaging system

Two-dimensional (2D) phase imaging system based on phase-shift interferometry (PSI) techniques can achieve a very high accuracy, but it has a degraded dynamic characteristic due to the inherent limitation of the PSI. Hence, the phase imaging system is incapable of obtaining real-time information pertaining to phase variations. To develop a label-free, high sensitivity, and dynamic bio-imaging system, a surface plasmon resonance (SPR) biosensing is combined with full-field heterodyne interferometry to develop a common-path full-field heterodyne SPR dynamic phase imager. The phase imager provides some advantages for biosensing such as label-free sensing, high sensitivity, high throughput, long-term stability, and dynamic capability. We build a 16×16 pixel photodiode array with a frame rate of up to 10 kHz as the 2D detector as opposed to a CCD camera with 30 Hz and employ an electro-optic modulator to generate a heterodyne light source. The multi-channel and real-time demodulation is calculated by utilizing a home-made digital signal processing-based lock-in amplifier. The SPR phase imager can detect refractive index changes better than 10-6 by testing the difference between nitrogen and argon gases, and will be used to analyze the biomolecular interaction on sensing surface with high throughput screening.

Study of cell adhesion and migration by using a plasmon-enhanced total internal reflection fluorescence microscope

Total internal reflection fluorescence microscopy (TIRFM) induces the evanescent field from an incident light with an incident angle greater than the critical angle selectively to excite fluorescent molecules on or near a surface. The TIRFM not only provides enhanced understanding of cellular function but also improves signal-to-noise ratio of detecting signal in real time. However, fluorescent emission need to be increased when a dynamic biomolecular image is requested at the frame rate of greater than 100 frames/s. Therefore, the fluorescent signal is enhanced via surface plasmons to match the requirements of better efficiency and larger quantity. In this study, a plasmon-enhanced TIRFM whose operation is based on the electromagnetic field enhancement via surface and particle plasmon effects offered by a nano-scalar silver thin film and particles has been presented. The developed microscopy has been successfully used in the real-time observation of the enhanced fluorescence from the thrombomodulin protein of a living cell membrane. The simulated and experimental results demonstrate that the plasmon-enhanced TIRFM can provide brighter living cell images through surface plasmon enhanced fluorescence.