Imaging of the cell surface interface using objective coupled widefield surface plasmon microscopy.

We report on the development and on the first use of the widefield surface plasmon (WSPR) microscope in the examination of the cell surface interface at submicron lateral resolutions. The microscope is Kohler illuminated and uses either a 1.45 numerical aperture (NA) oil immersion lens, or a 1.65 NA oil immersion lens to excite surface plasmons at the interface between a thin gold layer and a glass or sapphire cover slip. Like all surface plasmon microscope systems the WSPR has been proven in previous studies to also be capable of nanometric z-scale resolutions. In this study we used the system to image the interface between HaCaT cells and the gold layer. Imaging was performed in air using fixed samples and the 1.45 NA objective based system and also using live cells in culture media using the 1.65 NA based system. Imaging in air enabled the visualisation of high resolution and high-contrast submicron features identified by vinculin immunostaining as component of focal contacts and focal adhesions. In comparison, imaging in fluid enabled cell surface interfacial interactions to be tracked by time-lapse video WSPR microscopy. Our results indicate that the cell surface interface and thus cell signalling mechanisms may be readily interrogated in live cells without the use of labelling techniques.

Subpixel microscopic deformation analysis using correlation and artificial neural networks.

Microscopic deformation analysis has been performed using digital image correlation and artificial neural networks (ANNs). Cross-correlations of small image regions before and after deformation contain a peak, the position of which indicates the displacement to pixel accuracy. Subpixel resolution has been achieved here by nonintegral pixel shifting and by training ANNs to estimate the fractional part of the displacement. Results from displaced and thermally stressed microelectronic devices indicate these techniques can achieve comparable accuracies to other subpixel techniques and that the use of ANNs can facilitate very fast analysis without knowledge of the analytical form of the image correlation function.

High-precision tilt stage for the high-voltage electron microscope.

The high-voltage electron microscope is used to study thick samples (0.25 to several micrometers) to obtain three-dimensional information at ultrastructural resolution. Three-dimensional image reconstructions are often employed to extract, process and display this information. The sets of images used to form reconstructions must be recorded for precisely known specimen-beam orientations, especially if tomographic methods are employed. The design and operation of a precision (+/- 0.06 degrees) single-tilt stage to support this type of imaging is reported. All motions including two translations, height adjustment and tilting are accomplished via a single objective lens entry port. The specimen rod is supported on two rubber gaskets for vibration isolation, and motorized precision micrometers with encoder readouts for position monitoring drive the motions. The stage is stable to 0.6 nm for at least 16 s and is capable of tilt angles of +/- 70 degrees.

Design and operation of a differentially pumped environmental chamber for the HVEM.

A differentially pumped environmental chamber, or DIFPEC, has been developed for the AEI EM7 1.2 MV HVEM. The chamber is suitable for imaging and diffraction studies and is capable of operation at pressures approaching one atmosphere. Any mixture of gases can be used including water vapor supplied from an internal reservoir. Correct operation is demonstrated by measurements of temperature, pressure, and water consumption. High resolution (0.2 nm) diffraction patterns were recorded from unfixed, unstained, fully hydrated catalase crystals. This is good functional test of DIFPEC operation and specimen preparation because catalase disorders irreversibly of exposed to less than 95% relative humidity. High- and low-angle diffraction patterns were recorded from unfixed, unstained, fully hydrated rat hemoglobin crystals. All patterns were recorded using 10-4 C/cm2. Whole cell mounts prepared in different ways were imaged and show that 5.3 kPa of nitrogen gas has no detrimental effect on image contrast or resolution. The column vacuum and differentially pumped volume pressure were measured for a number of pumping configuration, as well as for several gases and pressures in the specimen volume. The pressure in the high vacuum portion of the objective lens gap is conductance-limited when the DIFPEC is in place.

Double-grating-structured light microscopy using plasmonic nanoparticle arrays.

Structured illumination increases the spatial bandwidth of optical microscopes. We demonstrate bandwidth extension using a physical grating placed close to the sample. This comprises an array of elongated nanoparticles, whose localized surface plasmon resonance is polarization dependent. By arranging the particle orientation to vary with position the grating can be moved by changing the input polarization. A projected optical grating provides an additional independent mechanism for bandwidth extension. Experimental results showing bandwidth improvement in one direction are presented, and the measures necessary to extend the technique for routine imaging are discussed.

Imaging performance of widefield solid immersion lens microscopy.

We investigate the performance of a widefield imaging system employing an aplanatic solid immersion lens. Off-axis imaging quality is examined theoretically at different radii and thicknesses of the aplanatic solid immersion lens. It is found that field curvature is the major aberration affecting the imaging quality. Aberrations are measured experimentally, and the results are in very good agreement with those obtained from simulations and demonstrate the situations where high quality images can be obtained with the aplanatic solid immersion lens.

Two-photon fluorescence surface wave microscopy.

This paper demonstrates the principle of two-photon surface wave microscopy with a view to applications on biological samples. We describe a modified scanning optical microscope, which uses specially prepared coverslips. These coverslips are designed to support the propagation of surface waves capable of large field enhancements. We also discuss the beam conditioning necessary to ensure efficient use of the available illumination. Two-photon surface wave fluorescent excitation is demonstrated on fluorescent nanospheres, demonstrating a point spread function width of approximately 220 nm at an illumination wavelength of 925 nm. The potential of non-linear surface wave excitation for both fluorescence and harmonic imaging microscopy is discussed.

Differential amplitude scanning optical microscope: theory and applications.

A differential amplitude scanning optical microscope is described. The operation of the system depends on the sinusoidal interrogation of the sample, at any scan coordinate, by moving the beam through a fraction of the focal spot on the object surface. The technique is capable of detecting variations in sample reflectivity down to 3 x10(-7) in a 10-Hz bandwidth. The image formation mechanism of the microscope is presented and applied to a number of important structures. Experimental results on grain structure of metals, surface integrity of polished diamond, and doped silicon wafers are presented. The microscope is well suited to the accurate measurement of linewidths. Theoretical and experimental results are presented to demonstrate this capability. It is further shown that the main limitations in providing accurate linewidth measurements are due to the accuracy of the scan coordinates rather than the optical aspects of the system.

Scanning differential phase contrast optical microscope: application to surface studies.

The characteristics and theory of operation of a new scanning differential phase contrast optical microscope are described, and a number of results are presented. High-contrast micrographs of a polished stainless steel sample are included, showing clearly the grain boundaries as well as some fine structure within the grains. Micrographs are also presented of natural diamonds both in polished and unpolished forms. In the former many polishing lines are visible, and in the latter one can clearly see a large number of stacking faults. Results on the study of monolayers of Langmuir-Blodgett films are also presented. The micrographs clearly show the boundaries as well as nonuniformities within the films. The ability of our system to image objects showing refractive-index variation is demonstrated by producing micrographs of an exposed but undeveloped photoresist film and a partially doped Si sample. In each case a qualitative comparison is made with the differential interference (Nomarski) micrograph of the same field of view.

Scanning optical microellipsometer for pure surface profiling.

We describe a scanning optical interferometer that can simultaneously perform ellipsometry measurements and thus provides a true surface profile. This is accomplished by projecting the back focal plane of the objective lens onto a CCD array. The measured phase differences between the p- and s-polarization components are converted, by using a specially developed algorithm, to optical phase changes caused by material variations. The compensation process is then applied to extract the true profile of the object surface. Experimental results obtained with the system are shown.

Single-probe-beam differential amplitude and phase-scanning interferometer.

We describe a common path differential amplitude and phase interferometer capable of measuring differential amplitude and phase responses simultaneously. The system uses a single probe beam that is imaged onto the detector plane; differentiation in any direction can be performed by the alteration of the position of the detectors. Experimental and theoretical results, which show excellent agreement, are presented to demonstrate how the transfer function of the system can be controlled to suit different application areas.

High-resolution wide-field surface plasmon microscopy.

This paper describes the application of a Köhler illuminated high-resolution wide-field microscope using surface plasmons to provide the image contrast. The response of the microscope to a grating structure in both the Fourier and the image planes is presented to demonstrate image formation by surface waves. The effect of spatial filtering in the back focal (Fourier) plane to enhance image contrast is described. We also discuss how the surface wave contrast mechanism affects the imaging performance of the microscope and discuss factors that can be expected to lead to even greater improvements in lateral resolution and sensitivity.

Optical V(z) for high-resolution 2pi surface plasmon microscopy.

Surface plasmons are electromagnetic surface waves whose k vectors are greater than that of free-space radiation. We excite surface plasmons by using an oil-immersion lens, which forms one arm of an interferometer. We demonstrate the way in which the characteristic output variation with defocus is determined by the propagation properties of the surface plasmons, which leads to diffraction-limited surface plasmon microscopy in the far field.

High-resolution scanning surface-plasmon microscopy.

Surface plasmons (SP's) are electromagnetic surface waves that propagate along the interface between conductors and dielectrics. The k vector of these waves is larger than the free-space wave vector. The importance of SP's lies in the fact that they are extremely sensitive to small changes in the dielectric properties of substances that are in contact with the conductors. This property means that SP's have many sensor applications; however, when they are used in microscopic applications the lateral resolution is limited to several micrometers. We discuss how this limit can be overcome by use of defocused high-numerical-aperture liquid-immersion objectives. We also present SP images that demonstrate a resolution comparable with that expected from high-numerical-aperture optical microscopes. Finally, we discuss how ultrahigh-numerical-aperture objectives with numerical apertures greater than 1.5 can be expected to have considerable influence on biological imaging.

Quantitative optical microscope with enhanced resolution using a pixelated liquid crystal spatial light modulator.

This paper presents a brief account of a novel optical microscope, which combines the advantages of two well-known techniques, namely phase contrast and phase stepping, to provide high contrast imaging and precision measurements. The inclusion of a programmable liquid crystal spatial light modulator provides for the phase stepping required, while also allowing flexibility for future improvements. The results shown reveal an important aspect of the system to facilitate quantitative sample measurements, with an enhancement of optical resolution compared with conventional optical imaging systems.

Ultrastable absolute-phase common-path optical profiler based on computer-generated holography.

A new scanning common-path interferometric profiler capable of absolute-phase measurement is described. The key element is a computer-generated hologram, which acts as the beam-splitting element. Unlike most absolute phase systems, it can be made entirely common path with respect to piston microphonics and is thus exceptionally stable. In addition to operating in scanning mode, the optical configuration permits simultaneous operation as a single-shot phase measuring interferometer and is thus capable of simultaneous form and texture measurements. The operation and stability of the scanning profiler are demonstrated experimentally.