Cells respond to forces through coordinated biochemical signaling cascades that originate from changes in single-molecule structure and dynamics and proceed to large-scale changes in cellular morphology and protein expression. To enable experiments that determine the molecular basis of mechanotransduction over these large time and length scales, we construct a confocal molecular dynamics microscope (CMDM). This system integrates total-internal-reflection fluorescence (TIRF), epifluorescence, differential interference contrast (DIC), and 3-D deconvolution imaging modalities with time-correlated single-photon counting (TCSPC) instrumentation and an optical trap. Some of the structures hypothesized to be involved in mechanotransduction are the glycocalyx, plasma membrane, actin cytoskeleton, focal adhesions, and cell-cell junctions. Through analysis of fluorescence fluctuations, single-molecule spectroscopic measurements [e.g., fluorescence correlation spectroscopy (FCS) and time-resolved fluorescence] can be correlated with these subcellular structures in adherent endothelial cells subjected to well-defined forces. We describe the construction of our multimodal microscope in detail and the calibrations necessary to define molecular dynamics in cell and model membranes. Finally, we discuss the potential applications of the system and its implications for the field of mechanotransduction.
Endothelial cells (ECs) convert mechanical stimuli into chemical signaling pathways to regulate their functions and properties. It is hypothesized that perturbation of cellular structures by force is accompanied by changes in molecular dynamics. In order to address these fundamental issues in mechanosensation and transduction, we have developed a hybrid multimodal microscopy - time-correlated single photon counting (TCSPC) spectroscopy system intended to determine time- and position dependent mechanically-induced changes in the dynamics of molecules in live cells as determined from fluorescence lifetimes and autocorrelation analysis (fluorescence correlation spectroscopy). Colocalization of cell-structures and mechanically-induced changes in molecular dynamics can be done in post-processing by comparing TCSPC data with 3-D models generated from total internal reflection fluorescence (TIRF), differential interference contrast (DIC), epifluorescence, and deconvolution. We present control experiments in which the precise location of the apical cell membrane with respect to a confocal probe is assessed using information obtainable only from TCSPC. Such positional accuracy of TCSPC measurements is essential to understanding the role of the membrane in mechanotransduction. We predict that TCSPC will become a useful method to obtain high temporal and spatial resolution information on localized mechanical phenomena in living endothelial cells. Such insight into mechanotransduction phenomenon may uncover the origins of mechanically-related diseases such as atherosclerosis.