Integrated biophotonics approach for noninvasive and multiscale studies of biomolecular and cellular biophysics.

Abstract

In the crowded cellular milieu, biological processes require coordinated intermolecular interactions, conformational changes, and molecular transport that span a wide range of spatial and temporal scales. This complexity requires an integrated, noninvasive, multiscale experimental approach. Here, we develop a multimodal fluorescence microspectroscopy system, integrated on a single platform, to gain information about molecular interactions and their dynamics with high spatio-temporal resolution. To demonstrate the versatility of our experimental approach, we use rhodamine 123-labeled mitochondria in breast cancer cells (Hs578T), verified using differential interference contrast (DIC) and fluorescence (confocal and two-photon) microscopy, as a model system. We develop an assay to convert fluorescence intensity to actual concentrations in intact, individual living cells, which contrasts with conventional biochemical techniques that require cell lysates. In this assay, we employ two-photon fluorescence lifetime imaging microscopy (FLIM) to quantify the fluorescence quantum yield variations found within individual cells. Functionally driven changes in cell environment, molecular conformation, and rotational diffusion are investigated using fluorescence polarization anisotropy imaging. Moreover, we quantify translational diffusion and chemical kinetics of large molecular assemblies using fluorescence correlation spectroscopy. Our integrated approach can be applied to a wide range of molecular and cellular processes, such as receptor-mediated signaling and metabolic activation.

• Authors: Qianru Yu, Michael Proia, Ahmed A Heikal
• Journal: Journal of biomedical optics (J Biomed Opt) 2008 Jul-Aug Vol. 13 Issue 4 Pg. 041315 ISSN: 1083-3668 [Print] United States
• PMID: 19021323 (Publication Type: Evaluation Study, Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, Non-P.H.S.)

Chemical References

• Neoplasm Proteins

Topics

• Breast Neoplasms (metabolism, pathology)
• Cell Line, Tumor
• Equipment Design
• Equipment Failure Analysis
• Humans
• Microscopy, Fluorescence (instrumentation)
• Microscopy, Polarization (instrumentation)
• Molecular Probe Techniques
• Neoplasm Proteins (metabolism)
• Spectrometry, Fluorescence (instrumentation)
• Systems Integration