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Subpixel digital image correlation has been applied to microscope images to analyze surface deformation. Nonintegral pixel shifting and successive approximation are used to calculate the subpixel element of the sample displacement without introducing systematic interpolation errors. Although in-plane displacement precision of better than 2% of a pixel, or < 15 nm at x10 magnification, is shown to be achievable, the use of even moderate numerical aperture microscope objectives render the technique sensitive to errors or variations in sample focusing. The magnitude of this effect is determined experimentally and a focus compensation method is described and demonstrated.

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.

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SummaryThis 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 ≈ 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.