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 live cell membranes via surface plasmon-enhanced fluorescence and phase microscopy.

This paper demonstrates the first combination for wide-field surface plasmon (SP) phase microscopy and SP-enhanced fluorescence microscopy to image living cells' contacts on the surface of a bio-substrate simultaneously. The phase microscopy with a phase-shift interferometry and common-path optical setup can provide high-sensitivity phase information in long-term stability. Simultaneously, the fluorescence microscopy with the enhancement of a local electromagnetic field can supply bright fluorescent images. The combined microscope imposes a high numerical aperture objective upon the excitation of surface plasmon through a silver film with a thickness of 30 nm. The developed SP microscope is successfully applied to the real-time bright observation of the transfected fluorescence of living cells localized near the cell membrane on the bio-substrate and the high-sensitivity phase image of the cell-substrate contacts at the same time.

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.

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 fluorescent images of living cell membranes. The proposed microscope with the help 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 a shorter fluorophore lifetime. In comparison with a 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. By combining the SP enhancement and two-photon excitation TIRF, the microscope has demonstrated it's capability for brighter and more contrasted fluorescence membrane images of living monkey kidney COS-7 fibroblasts transfected with an EYFP-MEM or EGFP-WOX1 construct.

Microfluidic systems integrated with two-dimensional surface plasmon resonance phase imaging systems for microarray immunoassay.

This study reports a microfluidic chip integrated with an arrayed immunoassay for surface plasmon resonance (SPR) phase imaging of specific bio-samples. The SPR phase imaging system uses a surface-sensitive optical technique to detect two-dimensional (2D) spatial phase variation caused by rabbit immunoglobulin G (IgG) adsorbed on an anti-rabbit IgG film. The microfluidic chip was fabricated by using micro-electro-mechanical-systems (MEMS) technology on glass and polydimethylsiloxane (PDMS) substrates to facilitate well-controlled and reproducible sample delivery and detection. Since SPR detection is very sensitive to temperature variation, a micromachine-based temperature control module comprising micro-heaters and temperature sensors was used to maintain a uniform temperature distribution inside the arrayed detection area with a variation of less than 0.3 degrees C. A self-assembled monolayer (SAM) technique was used to pattern the surface chemistry on a gold layer to immobilize anti-rabbit IgG on the modified substrates. The microfluidic chip is capable of transporting a precise amount of IgG solution by using micropumps/valves to the arrayed detection area such that highly sensitive, highly specific bio-sensing can be achieved. The developed microfluidic chips, which employed SPR phase imaging for immunoassay analysis, could successfully detect the interaction of anti-rabbit IgG and IgG. The interactions between immobilized anti-rabbit IgG and IgG with various concentrations have been measured. The detection limit is experimentally found to be 1 x 10(-4)mg/ml (0.67 nM). The specificity of the arrayed immunoassay was also explored. Experimental data show that only the rabbit IgG can be detected and the porcine IgG cannot be adsorbed. The developed microfluidic system is promising for various applications including medical diagnostics, microarray detection and observing protein-protein interactions.

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.

Surface plasmon resonance phase-shift interferometry: real-time DNA microarray hybridization analysis.

Surface plasmon resonance (SPR) phase-shift interferometry (PSI) is a novel technique which combines SPR and modified Mach-Zehnder PSI to measure the spatial phase variation caused by biomolecular interactions upon a sensing chip. The SPR-PSI imaging system offers high resolution and high-throughout screening capabilities for microarray DNA hybridization without the need for additional labeling, and provides valuable quantitative information. The SPR-PSI imaging system has an enhanced detection limit of 2.5 x 10(-7) refraction index change, a long-term phase stability of pi/100 in 30 min, and a spatial phase resolution of pi/300 with 100 x 100 microm2 detection area. This study successfully demonstrates the label-free observation of 15-mer DNA microarray.

Cell Adhesion on Micro-Structured Fibronectin Gradients Fabricated by Multiphoton Excited Photochemistry.

Concentration gradients of ECM proteins play active roles in many areas of cell biology including wound healing and metastasis. They may also form the basis of tissue engineering scaffolds, as these can direct cell adhesion and migration and promote new matrix synthesis. To better understand cell-matrix interactions on attractive gradients, we have used multiphoton excited (MPE) photochemistry to fabricate covalently linked micro-structured gradients from fibronectin (FN). The gradient design is comprised of a parallel series of individual linear gradients with overall dimensions of approximately 800 × 800 µm, where a linear dynamic range of nearly 10-fold in concentration was achieved. The adhesion dynamics of 3T3 fibroblasts were investigated, where the cell morphology and actin cytoskeleton became increasingly elongated and aligned with the direction of the gradient at increasing protein concentration. Moreover, the cell morphologies are distinct when adhered to regions of differing FN concentration but with similar topography. These results show that the fabrication approach allows investigating the roles of contact guidance and ECM cues on the cell-matrix interactions. We suggest this design overcomes some of the limitations with other fabrication methods, especially in terms of 3D patterning capabilities, and will serve as a new tool to study cell-matrix interactions.

Innovative antimicrobial susceptibility testing method using surface plasmon resonance.

Utilizing the ultra sensitivity of surface plasmon resonance (SPR) biosensor to examine drug resistance of bacteria was studied in this research. Susceptible and resistant strains of Escherichia coli JM109 to ampicillin and those of Staphylococcus epidermidis to tetracycline, served as a blind test, were examined. The bacteria adhered on the Au thin film was treated by the injection of antibiotic flow. The optical property change of the bacteria responded to antibiotics were recorded through SPR mechanism. As a result, the susceptible strain of E. coli generally revealed more than three times of SPR angle shift when compared to the resistant one; the susceptible strain of S. epidermidis revealed irregular SPR angle shift while the resistant strain kept the SPR angle almost unchanged. The new SPR method took less than 2h of antibiotic treatment time to complete the antimicrobial susceptibility test. Different from conventional applications of SPR, specific antibodies is not required in this method. As compared to the conventional assays, Kirby-Bauer disk diffusion and variations of broth microdilution usually take 1 day to weeks to issue the report. Using this SPR assay can greatly reduce the waiting period for laboratory tests, and can therefore benefit patients who need proper antibiotic treatments to control bacterial infections. The sensitivity of the SPR biosensor built for the application is around 1.4 x 10(-4) on the refractive index.