High Resolution Quantitative Angle-Scanning Widefield Surface Plasmon Microscopy.

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.

Time-dependent scattering of ultrathin gold film under potential perturbation.

Shifts of the plasmon scattering band of ultrathin gold films under the effect of dynamic applied potential were studied in single wavelength measurements. The effect on scattering of applied potential was ascribed to electronic charging and discharging of the gold film. Scattering transients in response to square-wave potential modulation had an exponential form which depended on the potential step width, the modulation frequency and the nature of the ions. The presence of an AC signal component induced by ±10 mV potential modulated at 2 kHz indicated the capability of very thin gold film to respond to high frequency voltage.

A reconfigurable real-time compressive-sampling camera for biological applications.

Many applications in biology, such as long-term functional imaging of neural and cardiac systems, require continuous high-speed imaging. This is typically not possible, however, using commercially available systems. The frame rate and the recording time of high-speed cameras are limited by the digitization rate and the capacity of on-camera memory. Further restrictions are often imposed by the limited bandwidth of the data link to the host computer. Even if the system bandwidth is not a limiting factor, continuous high-speed acquisition results in very large volumes of data that are difficult to handle, particularly when real-time analysis is required. In response to this issue many cameras allow a predetermined, rectangular region of interest (ROI) to be sampled, however this approach lacks flexibility and is blind to the image region outside of the ROI. We have addressed this problem by building a camera system using a randomly-addressable CMOS sensor. The camera has a low bandwidth, but is able to capture continuous high-speed images of an arbitrarily defined ROI, using most of the available bandwidth, while simultaneously acquiring low-speed, full frame images using the remaining bandwidth. In addition, the camera is able to use the full-frame information to recalculate the positions of targets and update the high-speed ROIs without interrupting acquisition. In this way the camera is capable of imaging moving targets at high-speed while simultaneously imaging the whole frame at a lower speed. We have used this camera system to monitor the heartbeat and blood cell flow of a water flea (Daphnia) at frame rates in excess of 1500 fps.

Morphology-dependent voltage sensitivity of a gold nanostructure.

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.

Wide-field high-resolution structured illumination solid immersion fluorescence microscopy.

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.

Polarization modulation thermal lens microscopy for imaging the orientation of non-spherical nanoparticles.

In this paper a far field optical technique we call polarization modulation thermal lens microscopy (PM-TLM) is used for imaging the orientation and dichroism of non-spherical nanoparticles. In PM-TLM, the polarization state of a pump beam is periodically modulated which in turn causes morphology related intensity fluctuations in a continuous probe beam, thus allowing high signal to noise ratio detection with using lock-in amplification. Since PM-TLM uses nanoparticle absorption as the contrast mechanism, it may be used to detect and image nanoparticles of far smaller dimensions than can be observed by conventional dark field optical microscopy. The technique, its implementation and experiment results are presented.

Multichannel, time-resolved picosecond laser ultrasound imaging and spectroscopy with custom complementary metal-oxide-semiconductor detector.

This paper presents a multichannel, time-resolved picosecond laser ultrasound system that uses a custom complementary metal-oxide-semiconductor linear array detector. This novel sensor allows parallel phase-sensitive detection of very low contrast modulated signals with performance in each channel comparable to that of a discrete photodiode and a lock-in amplifier. Application of the instrument is demonstrated by parallelizing spatial measurements to produce two-dimensional thickness maps on a layered sample, and spectroscopic parallelization is demonstrated by presenting the measured Brillouin oscillations from a gallium arsenide wafer. This paper demonstrates the significant advantages of our approach to pump probe systems, especially picosecond ultrasonics.

Quantitative studies of the interactions of metalloproteins with gold nanoparticles: identification of dominant properties of the protein that underlies the spectral changes.

The interaction of cytochrome C and a number of its components such as the apo protein, heme and a coordinated iron with gold nanospheres, has been investigated. The role of the heme group and its effect on the observed spectroscopic properties following binding of cytochrome C to the gold surface has been evaluated. Binding of the heme group directly to the gold is not observed, but the presence of the heme group and its effect on the interaction with the metal surface is shown to be influential. Other factors such as the metal oxidation state and the metal-free heme are also studied. A comparison to serum albumin binding as a nonmetallic protein provides further insight into the interaction characteristics.

Stochastic transfer function: application to fluorescence microscopy.

We develop the concept of the stochastic transfer function (STF) and its application to high-resolution fluorescence microscopy. The STF is directly related to the conventional optical transfer functions but incorporates a probability density function at each spatial frequency. The mean of the STF yields the conventional transfer function; the variance of the STF gives a measure of the noise associated at each spatial frequency. The STF thus provides a more complete picture of the microscope performance in comparison with conventional transfer functions. Here we present the STF for a wide-field fluorescent microscope using both Monte Carlo simulation and probability theory.

Stochastic transfer function for structured illumination microscopy.

The stochastic transfer function extends the concept of the conventional transfer function by incorporating noise statistics, thus giving a measure of the signal-to-noise ratio at each spatial frequency. This provides a convenient and standardized metric to assess the trade-off in terms of spatial frequency bandwidth and signal-to-noise ratio for a diverse range of resolution enhancement techniques. We apply this concept to structured illumination microscopy and compare its noise performance as a function of frequency with the conventional wide-field fluorescent microscope. The result suggests how a hybrid algorithm further improves the photon efficiency of the structured illumination technique.

Resolution in structured illumination microscopy: a probabilistic approach.

Structured illumination can be employed to extend the lateral resolution of wide-field fluorescence microscopy. Since a structured illumination microscopy image is reconstructed from a series of several acquired images, we develop a modified formulation of the imaging response of the microscope and a probabilistic analysis to assess the resolution performance. We use this model to compare the fluorescence imaging performance of structured illumination techniques to confocal microscopy. Specifically, we examine the trade-off between achievable lateral resolution and signal-to-noise ratio when photon shot noise is dominant. We conclude that for a given photon budget, structured illumination invariably achieves better lateral resolution than confocal microscopy.

Surface-plasmon microscopy with a two-piece solid immersion lens: bright and dark fields.

We report bright-field and dark-field surface-plasmon imaging using a modified solid immersion lens and a commercial objective of moderate NA in the epi configuration. The contrast and resolution are extremely good, giving well-resolved images of protein monolayers both in air and in water. We also describe a two-part solid immersion lens that allows the sample to be moved without degrading the image quality in any observable way. The merits of the two-part lens are discussed and compared to commercially available microscope objectives. Finally, we introduce a simple Green's function model that illustrates the key features of both bright-field and dark-field surface-plasmon imaging.

Full-field heterodyne interference microscope with spatially incoherent illumination.

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.

Remote photoacoustic detection of liquid contamination of a surface.

A method for the remote detection and identification of liquid chemicals at ranges of tens of meters is presented. The technique uses pulsed indirect photoacoustic spectroscopy in the 10-microm wavelength region. Enhanced sensitivity is brought about by three main system developments: (1) increased laser-pulse energy (150 microJ/pulse), leading to increased strength of the generated photoacoustic signal; (2) increased microphone sensitivity and improved directionality by the use of a 60-cm-diameter parabolic dish; and (3) signal processing that allows improved discrimination of the signal from noise levels through prior knowledge of the pulse shape and pulse-repetition frequency. The practical aspects of applying the technique in a field environment are briefly examined, and possible applications of this technique are discussed.

Characterization of layered scattering media using polarized light measurements and neural networks.

Measurements of the spatial distributions of polarized light backscattered from a two-layer scattering medium are used to train a neural network. We investigated whether the absorption coefficients and thickness of the layer can be determined when the scattering properties are known. When determining the absorption of the upper layer or the layer's thickness, polarized light measurements provide better performance than unpolarized measurements, demonstrating the sensitivity of polarized light to superficial tissue. Determination of the lower layer's absorption coefficient is not improved by polarized light measurements. Prior knowledge of the tissue under investigation is also beneficial because errors are reduced if the range of absorption or thickness is restricted.