Hyperspectral image correlation for monitoring membrane protein dynamics in living cells

Ryan W. Davis; Bryan Carson; Howland D. T. Jones; Michael B. Sinclair

doi:10.1117/12.807743

3 March 2009 Hyperspectral image correlation for monitoring membrane protein dynamics in living cells

Ryan W. Davis, Bryan Carson, Howland D. T. Jones, Michael B. Sinclair

Proceedings Volume 7184, Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XVI; 71840J (2009) https://doi.org/10.1117/12.807743
Event: SPIE BiOS, 2009, San Jose, California, United States

Abstract

Temporal image correlation provides a powerful fluorescence technique for measuring several biologically relevant parameters of molecules in living cells. These parameters include, but are not limited to local concentrations, diffusion dynamics, and aggregation states of biomolecules. However, the complex cellular environment presents several limitations, precluding high quantitative accuracy and constraining biological implementation. In order to address these issues, high speed spectral imaging was employed to compare the results of image correlation from spectrally unmixed and virtually implemented fluorescence emission filters. Of particular interest in this study is the impact of cellular autofluorescence, which is ubiquitous in fluorescence imaging of cells and tissues. Using traditional instrumentation, corrections for autofluorescence are commonly estimated as a static offset collected from a separate control specimen. While this may be sufficient in highly homogenous regions of interest, the low analyte concentrations requisite to fluctuation-based methods result in the potential for unbounded error resulting from spectral cross-talk between local autofluorescence inhomogeneities and the fluorescence signal of interest. Thus we demonstrate the importance of accurate autofluorescence characterization and discuss potential corrections using a case study focusing on fluorescence confocal spectral imaging of immune cells before and after stimulation with lipopolysaccheride (LPS). In these experiments, binding of LPS to the membrane receptor, YFP-TLR4, is observed to result in initiation of the immune response characterized by altered receptor diffusion dynamics and apparent heterogeneous aggregation states. In addition to characterizing errors resulting from autofluorescence spectral bleed-through, we present data leading to a deeper understanding of the molecular dynamics of the immune response and suggest hypotheses for future work utilizing hyperspectrally enabled multi-label fluorescence studies on this system of high biological import.

Citation Download Citation

Ryan W. Davis, Bryan Carson, Howland D. T. Jones, and Michael B. Sinclair "Hyperspectral image correlation for monitoring membrane protein dynamics in living cells", Proc. SPIE 7184, Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XVI, 71840J (3 March 2009); https://doi.org/10.1117/12.807743

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