Fluorescence fluctuation analysis of mixed chromophores from a line-scanning hyperspectral imaging system

Ryan W. Davis; Jesse S. Aaron; Susan L. Rempe; Jerilyn A. Timlin

doi:10.1117/12.842294

17 February 2010 Fluorescence fluctuation analysis of mixed chromophores from a line-scanning hyperspectral imaging system

Ryan W. Davis, Jesse S. Aaron, Susan L. Rempe, Jerilyn A. Timlin

Author Affiliations +

Proceedings Volume 7570, Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XVII; 757002 (2010) https://doi.org/10.1117/12.842294
Event: SPIE BiOS, 2010, San Francisco, California, United States

Abstract

Fluorescence fluctuation analysis of dilute biomolecules can provide a powerful method for fast and accurate determination of diffusion dynamics, local concentrations, and aggregation states in complex environments. However, spectral overlap among multiple exogenous and endogenous fluorescent species, photobleaching, and background inhomogeneities can compromise quantitative accuracy and constrain useful biological implementation of this analytical strategy in real systems. In order to better understand these limitations and expand the utility of fluctuation correlation methods, spatiotemporal fluorescence correlation analysis was performed on spectrally resolved line scanned images of modeled and real data from mixed fluorescent nanospheres in a synthetic gel matrix. It was found that collecting images at a pixel sampling regime optimal for spectral imaging provides a method for calibration and subsequent temporal correlation analysis which is insensitive to spectral mixing, spatial inhomogeneity, and photobleaching. In these analyses, preprocessing with multivariate curve resolution (MCR) provided the local concentrations of each spectral component in the images, thus facilitating correlation analysis of each component individually. This approach allowed quantitative removal of background signals and showed dramatically improved quantitative results compared to a hypothetical system employing idealized filters and multi-parameter fitting routines.

Citation Download Citation

Ryan W. Davis, Jesse S. Aaron, Susan L. Rempe, and Jerilyn A. Timlin "Fluorescence fluctuation analysis of mixed chromophores from a line-scanning hyperspectral imaging system", Proc. SPIE 7570, Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XVII, 757002 (17 February 2010); https://doi.org/10.1117/12.842294

ACCESS THE FULL ARTICLE

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