Gold nanostructures of various morphologies, including nanospheres, nanorods, nanoprisms, and thin films, were immobilized on ITO-coated coverslips in order to investigate the response of their scattering to potential. Shifts in the plasmon band obtained by potential-modulated spectroscopic imaging indicated that the voltage sensitivity of the gold nanostructure is dependent on its morphology, with nanospheres exhibiting the lowest sensitivity and ultrathin gold films exhibiting the highest. The effects of potential on gold nanoparticles are in qualitative agreement with Mie and Gans' theories in which the shift of the gold plasma frequency is due to the charging-discharging of the nanoparticles.

A heterodyne interference microscope arrangement for full-field imaging is described. The reference and object beams are formed with highly correlated, time-varying laser speckle patterns. The speckle illumination confers a confocal transfer function to the system, and by temporal averaging, the coherence noise that often degrades coherent full-field microscope images is suppressed. The microscope described is similar to a Linnik-type microscope and allows the use of high-numerical-aperture objective lenses, but the temporal coherence of the illumination permits the use of a low-power achromatic doublet in the reference arm. The use of a doublet simplifies alignment of the microscope and can reduce the cost. Preliminary results are presented that demonstrate full-field surface height precision of 1 nm rms.

Key words. Drug delivery, endocytosis, evanescent wave microscopy, imaging systems, live-cell imaging, medical and biological imaging, microscopy, total internal reflection fluorescence. SummaryThis paper presents a simple, high-resolution, non-fluorescent imaging technique called total internal reflection microscopy (TIRM) and demonstrates its potential application to realtime imaging of live cellular events. In addition, a novel instrument is introduced that combines the simplicity of TIRM with the specificity afforded by dual-colour total internal reflection fluorescence (TIRF) microscopy and allows sequential imaging with the two modalities. The key design considerations necessary to apply these imaging modes in a single instrument are discussed. The application of TIRM alone yielded high-resolution live images of cell adherence to a poly-L -lysine modified substrate, whereby fine cellular structures are imaged. Non-fluorescent imaging of the uptake of sub-micron-sized polymeric particles by live cells is also demonstrated. Finally, images of fluorescently labelled cells were obtained in TIRF mode, sequentially to images obtained of the same cell in TIRM mode. Visual information gained using TIRF is compared with TIRM to demonstrate that the level of cell structure information obtainable with our total internal reflection microscope is comparable with the TIRF technique.

We describe the construction of a prismless widefield surface plasmon microscope; this has been applied to imaging of the interactions of protein and antibodies in aqueous media. The illumination angle of spatially incoherent diffuse laser illumination was controlled with an amplitude spatial light modulator placed in a conjugate back focal plane to allow dynamic control of the illumination angle. Quantitative surface plasmon microscopy images with high spatial resolution were acquired by post-processing a series of images obtained as a function of illumination angle. Experimental results are presented showing spatially and temporally resolved binding of a protein to a ligand. We also show theoretical results calculated by vector diffraction theory that accurately predict the response of the microscope on a spatially varying sample thus allowing proper quantification and interpretation of the experimental results.

The use of aplanatic solid immersion lenses (ASILs) made of high-refractive-index optical materials provides a route to wide-field high-resolution optical microscopy. Structured illumination microscopy (SIM) can double the spatial bandwidth of a microscope to also achieve high-resolution imaging. We investigate the combination of ASILs and SIM in fluorescence microscopy, which we call structured illumination solid immersion fluorescence microscopy (SISIM), to pursue a microscopic system with very large NA and high lateral resolution. We demonstrate that the combination can produce a wide-field high-resolution microscopic system with bandwidth corresponding to an NA of 3. Future developments of the SISIM system to make it achieve even higher resolution are proposed.