Optical fibers can deliver light to, and collect it from, regions deep in tissue. However, reported illumination and fluorescence collection volumes adjacent to the fiber tip have been inconsistent, and systematic data on this topic are not available. Illumination and fluorescence collection profiles were characterized with high spatial resolution for different optical fibers in tissue and various fluids using two-photon flash photolysis and excitation. We confirm that illumination and fluorescence collection volumes for optical fibers are near identical. Collection volume is determined by the core dimensions and numerical aperture (NA) of the fiber and the scattering properties of the medium. For a multimode optical fiber with 100 µm core diam and NA=0.22, 80% of the total fluorescence is collected from a depth of 170 µm in tissue and 465 µm in nonscattering fluid. A semiempirical mathematical description of photon flux adjacent to the fiber tip was also developed and validated. This was used to quantify the extent of temporal blurring associated with propagation of a wavefront of altered fluorescence emission across the region addressed by fiber optic probes. We provide information that will facilitate the design of optical probes for tissue imaging or therapeutic applications.
The availability of rapid and specific biosensors is of great importance for many areas of biomedical
research and modern biotechnology. This includes a need for DNA sensors where the progress of molecular
biology demands routine detection of minute concentrations of specific gene fragments. A promising
alternative approach to traditional DNA essays utilizes novel smart materials, including conducting
polymers and nanostructured materials such as quantum dots. We have constructed a number of DNA
sensors based on smart materials that allow rapid one-step detection of unlabeled DNA fragments with high
specificity. These sensors are based on functionalized conducting polymers derived from polypyrrole (PPy)
and poly(p-phenylenevinylene) (PPV). PPy based sensors provide intrinsic electrical readout via cyclic
voltammetry and electrochemical impedance spectroscopy. The performance of these sensors is compared
to a novel self-assembled monolayer-PNA construct on a gold electrode. Characterization of the novel PNA
based sensor shows that it has comparable performance to the PPy based sensors and can also be read out
effectively using AC cyclic voltammetry. Complementary to such solid substrate sensors we have
developed a novel optical DNA essay based on a new PPV derived cationic conducting polymer. DNA
detection in this essay results from sample dependent fluorescence resonance energy transfer changes
between the cationic conducting polymer and Cy3 labeled probe oligonucleotides. As an alternative to such
fluorochrome based sensors we discuss the use of inorganic nanocrystals ('quantum dots') and present data
from water soluble CdTe quantum dots synthesized in an aqueous environment.
Two-photon excitation (TPE) via a microscope objective lens produces a spatially confined excitation volume where UV-excited caged molecules may be broken (uncaged) to release active products. We describe an optical system that creates a stationary parfocal TPE uncaging spot on the stage of a conventional confocal microscope. With this system, we have examined the ability of two dyes to track microscopic calcium changes produced by TPE photolysis of DM-nitrophen. We find that, even when EGTA is used with a low affinity indicator, the dye signals are complicated by diffusion of both indicator-Ca complex and CaEGTA to produce a signal that does not simply report the spatial dimensions of the calcium release site. In addition, the time course of calcium release is poorly reported. This suggests that considerable caution must be applied to the interpretation of spatially resolved calcium signals inside cells. We have also used TPE of CMND-caged fluorescein to measure the rate of fluorescein production in test solution (2500 s-1) as well as the diffusion of fluorescein in drops of solution and within and between between eye lens fiber cells. While diffusion of uncaged fluorescein was about an order of magnitude slower inside fiber cells than in aequeous solution, slower diffusion between cells could also be detected and could be explained by the gap junctions joining the cells behaving as a barrier to diffusion. By using a computer model, parameter fits to experimental data gave estimates for both intracellular and intercellular diffusion coefficients. From this analysis, the gap junctions in eye lens fiber cells permit exchange of low molecular weight compounds between cells at about 0.4% of the rate of free diffusion.