Dr. John G. Williams Profile

Dr. John G. Williams

Principal Scientist at LI-COR Biosciences

SPIE Involvement:

Author

Publications (3)

This will count as one of your downloads.

You will have access to both the presentation and article (if available).

DOWNLOAD NOW

This content is available for download via your institution's subscription. To access this item, please sign in to your personal account.

Email or Username Forgot your username?

Password Forgot your password?

Show

Keep me signed in

No SPIE account? Create an account

Proceedings Article | 22 September 2007 Paper

Analysis of metal enhanced fluorescence on near-IR fluorophores

Jon Anderson, Mark Griffiths, John Williams, Daniel Grone, Don Lamb, Vince Boveia

Proceedings Volume 6641, 664108 (2007) https://doi.org/10.1117/12.731628

KEYWORDS: Luminescence, Silver, Glasses, Metals, Nanostructures, Proteins, Molecular biology, Signal detection, Visible radiation, Imaging systems

Read Abstract +

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.

Proceedings Article | 24 April 2001 Paper

Imaging of single-chromophore molecules in aqueous solution near a fused-silica interface

Lloyd Davis, Wesley Parker, David Ball, John Williams, Greg Bashford, Pamela Sheaff, Robert Eckles, Don Lamb, Lyle Middendorf

Proceedings Volume 4262, (2001) https://doi.org/10.1117/12.424568

KEYWORDS: Molecules, Prisms, Luminescence, Cameras, Molecular lasers, Microscopes, Silica, Diffusion, CCD cameras, Raman spectroscopy

Read Abstract +

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.

Proceedings Article | 15 January 1999 Paper

Computer simulation of gene detection without PCR by single molecule detection

Lloyd Davis, John Williams, Don Lamb

Proceedings Volume 3570, (1999) https://doi.org/10.1117/12.336943

KEYWORDS: Molecules, Molecular lasers, Luminescence, Computer simulations, Monte Carlo methods, Quantum efficiency, Photodetectors, Infrared radiation, Semiconductor lasers, Diffusion

Read Abstract +

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.

My Library

You currently do not have any folders to save your paper to! Create a new folder below.

Folder Name

Folder Description

View contact details

UPDATE YOUR PROFILE

Is this your profile? Update it now.

Sign into your SPIE.org account

Don’t have a profile and want one?

Create an account on SPIE.org

Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks. You are receiving this notice because your organization may not have SPIE eBooks access.*

*Shibboleth/Open Athens users─please sign in to access your institution's subscriptions.

To obtain this item, you may purchase the complete book in print or electronic format on SPIE.org.

ORGANIZATIONAL
Sign in with credentials provided by your organization.

Organizational Username

Organizational Password

Show/Hide Password

INSTITUTIONAL
Select your institution to access the SPIE Digital Library.

SELECT YOUR INSTITUTION

PERSONAL
Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.

PERSONAL SIGN IN

No SPIE Account? Create one

PURCHASE THIS CONTENT

SUBSCRIBE TO DIGITAL LIBRARY

50 downloads per 1-year subscription

Members: $195

Non-members: $335 ADD TO CART

25 downloads per 1 - year subscription

Members: $145

Non-members: $250 ADD TO CART

PURCHASE SINGLE ARTICLE

Includes PDF, HTML & Video, when available

Members:

Non-members: ADD TO CART

Keywords/Phrases

Search In:

Publication Years