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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.

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.

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.

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.