Fluorescence is a highly sensitive, precise, and convenient detection technique that is widely used in chemistry,
molecular biology and clinical laboratories. Fluorescence in the near-IR (700 - 900 nm) offers higher molar absorptivity
and significantly lower background signals from scatter than those generated by visible wavelength excitation. The
advantageous characteristics of near-IR fluorescence, primarily the reduced background signals, make this region of the
spectrum ideal for enhancement by metal nanostructures. Though multiple groups have successfully demonstrated metal
enhanced fluorescence, there remain several challenges in transferring this technology from the research stage to the
commercial stage. Using a LI-COR Odyssey® Infrared Imaging System, we quantitatively analyzed the effects of silver
particle geometries, including size, shape, and density of metal nanostructures, on the fluorescence enhancement of
Near-IR fluorophores. Using silver island film coated glass slides, we were able to obtain an 18-fold enhancement of
IRDye®700 and a 15-fold enhancement of IRDye®800 labeled DNA oligos over dye on plain glass. We further analyzed
the silver-coated glass surfaces for enhancement reproducibility and linearity. We demonstrated that the metal enhanced
emissions remained reproducible across a slide surface, and remained linear over several orders of magnitude. Finally,
using a highly quenched labeled protein, we were able to show an enhancement and release of the quenched
fluorescence, generating a 40-fold enhancement in the fluorescence emissions when spotted on a silver nanostructure
coated glass slide. Generating silver nanostructure coated slides that enhance fluorescence while maintaining linearity
and reproducibility will provide a class of new tools benefiting molecular biologists.
Single molecules of unconjugated Bodipy-Texas Red (BTR), BTR-dimer, and BTR conjugated to cysteine, in aqueous solutions are imaged using total-internal-reflection excitation and through-sample collection of fluorescence onto an intensified CCD camera, or a back-illuminated frame transfer CCD. The sample excitation is provided by the beam from a continuous-wave krypton ion laser, or a synchronously-pumped dye laser, operating at 568 nm. In order to essentially freeze molecular motion due to diffusion and thereby enhance image contrast, the laser beam is first passed through a mechanical shutter, which yields a 3-millisecond laser exposure for each camera frame. The laser beam strikes the fused-silica/sample interface at an angle exceeding the critical angle by about 1 degree. The resultant evanescent wave penetrates into the sample a depth of approximately 0.3 microns. Fluorescence from the thin plane of illumination is then imaged onto the camera by a water immersion apochromat (NA 1.2, WD 0.2mm). A Raman notch filter blocks Rayleigh and specular laser scatter and a band-pass-filter blocks most Raman light scatter that originates from the solvent. Single molecules that have diffused into the evanescent zone at the time of laser exposure yield near-diffraction-limited Airy disk images with diameters of ~5 pixels. While most molecules diffuse out of the evanescent zone before the next laser exposure, stationary or slowly moving molecules persisting over several frames, and blinking of such molecules are occasionally observed.
Pioneer Hi-Bred is developing a low-cost method for rapid screening of DNA, for use in research on elite crop seed genetics. Unamplified genomic DNA with the requisite base sequence is simultaneously labeled by two different colored fluorescent probes, which hybridize near the selected gene. Dual-channel single molecule detection (SMD) within a flow cell, then provides a sensitive and specific assay for the gene. The technique has been demonstrated using frequency- doubled Nd:YAG laser excitation of two visible-wavelength dyes. A prototype instrument employing infrared fluorophores and laser diodes for excitation has been developed. Here, we report results from a Monte Carlo simulation of the new instrument, in which experimentally determined photophysical parameters for candidate infrared dyes are used for parametric studies of experimental operating conditions. Fluorophore photostability is found to be a key factor in determining the instrument sensitivity. Most infrared dyes have poor photostability, resulting in inefficient SMD. However, the normalized cross-correlation function of the photon signals from each of the two channels can still yield a discernable peak, provided that the concentration of dual- labeled molecules is sufficiently high. Further, for low concentrations, processing of the two photon streams with Gaussian -weighted sliding sum digital filters and selection of simultaneously occurring peaks can also provide a sensitive indicator of the presence of dual-labeled molecules, although accidental coincidences must be considered in the interpretation of results.
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