Pterin detection using surface-enhanced Raman spectroscopy incorporating a straightforward silver colloid-based synthesis technique.

Optical techniques toward the realization of sensitive and selective biosensing platforms have received considerable attention in recent times. Techniques based on interferometry, surface plasmon resonance, and waveguides have all proved popular, while spectroscopy in particular offers much potential. Raman spectroscopy is an information-rich technique in which the vibrational frequencies reveal much about the structure of a compound, but it is a weak process and offers poor sensitivity. In response to this problem, surface-enhanced Raman scattering (SERS) has received much attention, due to significant increases in sensitivity instigated by bringing the sample into contact with an enhancing substrate. Here we discuss a facile and rapid technique for the detection of pterins using SERS-active colloidal silver suspensions. Pterins are a family of biological compounds that are employed in nature in color pigmentation and as facilitators in metabolic pathways. In this work, small volumes of xanthopterin, isoxanthopterin, and 7,8-dihydrobiopterin have been examined while adsorbed to silver colloids. Limits of detection have been examined for both xanthopterin and isoxanthopterin using a 10-s exposure to a 12 mW 532 nm laser, which, while showing a trade-off between scan time and signal intensity, still provides the opportunity for the investigation of simultaneous detection of both pterins in solution.

The photoluminescent lifetime of polyelectrolytes in thin films formed via layer by layer self-assembly.

We present results on luminescence lifetime studies of thin multilayer films of polyelectrolyte molecules produced via layer by layer (LbL) electrostatic assembly. We found that, in contrast to common assumptions, LbL films show measurable photoluminescent lifetimes with an average value of 6 ns. Scanning fluorescence lifetime imaging microscopy studies combined with steady-state photoluminescence measurements imply that this lifetime may be due to aggregation of polyelectrolyte molecules during preparation of LbL films. This conclusion has been further confirmed by atomic force microscopy (AFM). AFM images clearly show the presence of 100-200 nm high aggregates on the surface of these films. This aggregation of polyelectrolyte molecules contributes significantly to the experimentally detected luminescence decays of any light-emitting samples attached to LbL film, especially in a single molecule detection regime. To demonstrate this effect we compare photoluminescence lifetime results for CdTe quantum dots deposited on the surface of LbL polyelectrolyte films.

Confocal microscopy using variable-focal-length microlenses and an optical fiber bundle.

The use of variable-focal-length (VFL) microlenses can provide a way to axially scan the foci across a sample by electronic control. We demonstrate an approach to coupling VFL microlenses individually to a fiber bundle as a way to create a high-throughput aperture array with a controllable aperture pattern. It would potentially be applied in real-time confocal imaging in vivo for biological specimens. The VFL microlenses that we used consist of a liquid-crystal film sandwiched between a pair of conductive substrates for which one has a hole-patterned electrode. One obtains the variation of the focal length by changing the applied voltage. The fiber bundle has been characterized by coupling with both coherent and incoherent light sources. We further demonstrate the use of a VFL microlens array in combination with the fiber bundle to build up a confocal system. The axial response of the confocal system has been measured without mechanical movement of the sample or the objective, and the FWHM is estimated to be approximately 16 microm, with asymmetric sidelobes.

Three-dimensional imaging of microspheres with confocal and conventional polarization microscopes.

We experimentally studied the three-dimensional imaging of the microspheres by using confocal and conventional scanning polarization microscopes. Because of the field amplitude averaging effect of the confocal system, the polarization of the detected signals is mainly parallel to the initial polarization. As a result, the signal intensity from the microspheres in the confocal polarization microscope with a crossed analyzer was found to be weaker than that in the conventional system. Based on a vector approach that takes the polarization into account and on the image formations of the two systems, theoretical expressions are given that agree well with the experimental results.

Effects of source coherence and aperture array geometry on optical sectioning strength in direct-view microscopy.

Laser sources offer a possible solution to the problem of low light throughput in direct-view microscopes (DVMs). However, coherent source DVMs have been shown to suffer from problems such as increased sidelobes in the depth response because of coherent cross talk between neighboring apertures. We explore theoretically how source coherence affects the depth responses of DVMs by employing various aperture spacings and number of apertures. We show that, contrary to expectation, closely spaced apertures can result in decreased full width at half-maximum of the depth response curve. We explain this as an effect of destructive interference when cross talk between neighboring apertures occurs. Using apertures arranged in a square grid as an example, we move on to show that the use of aperture arrays that consist of regularly arranged apertures can accentuate the problematic sidelobes of the depth response. We show that arranging pinholes in a rectangular grid rather than a square grid can improve the optical sectioning strength significantly. Finally, by examination of the depth responses corresponding to the infinite-pinhole-array limit, we make some general statements about source coherence and the characteristics of arrays that are likely to perform well.