The objective of this work is to develop a new approach to multichannel fluorescence spectroscopy that will result in extended sensitivity for a number of imaging applications in biomedical research, including high-speed multiplexed read-out of bio-chips, rapid DNA sequencing, fluorescence lifetime imaging microscopy, and cell imaging experiments that require separation of fluorescent labels with overlapping excitation and/or emission spectra, such as green fluorescent protein and its variations.
The aims i nclude implementation of a scanning confocal microscope with multiple excitation wavelengths, multiple emission wavelength bands, and time-resolved single-photon detection, and also implementation of statistically efficient maximum-likelihood based methods for the resolution of the resultant multi-channel data sets into the fractional components corresponding to each fluorophore present, and background. Numerical methods will be used to solve the analytically intractable maximum-likelihood equations to determine the component fractions and their errors and co-dependence. Analysis software will be made available for internet download. Also, a procedure will be developed for the optimal selection of the excitation wavelengths and emission bands for given fluorophores and experimental conditions. The maximum-likelihood based approach will be evaluated using experimental test data. Monte Carlo simulations will also be developed to validate the new approach using the spectra of commonly-used fluorophores and to determine its capabilities in terms of the accuracy (lack of bias), statistical precision, and covariance of parameters for given signal and background levels, particularly for photon-starved imaging applications. The performance range of the maximum-likelihood based approached will be compared with that of existing least-squares curve-fitting-based """"""""linear un-mixing"""""""", which is expected to become statistically invalid for low-count data sets.

Agency
National Institute of Health (NIH)
Institute
National Institute of Biomedical Imaging and Bioengineering (NIBIB)
Type
Small Research Grants (R03)
Project #
5R03EB004586-02
Application #
7097965
Study Section
Special Emphasis Panel (ZRG1-BST-A (10))
Program Officer
Korte, Brenda
Project Start
2005-08-01
Project End
2008-07-31
Budget Start
2006-08-01
Budget End
2008-07-31
Support Year
2
Fiscal Year
2006
Total Cost
$73,384
Indirect Cost
Name
University of Tennessee Space Institute
Department
Type
Organized Research Units
DUNS #
085834182
City
Tullahoma
State
TN
Country
United States
Zip Code
37388
Davis, Lloyd M; Shen, Guoqing (2006) Accounting for triplet and saturation effects in FCS measurements. Curr Pharm Biotechnol 7:287-301