Integrated multimodal microscopy, time-resolved fluorescence, and optical-trap rheometry: toward single molecule mechanobiology.
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.
Gullapalli
Ramachandra R
RR
The Pennsylvania State University, Department of Bioengineering, 205 Hallowell Building, University Park, Pennsylvania 16802, USA.
Tabouillot
Tristan
T
Mathura
Rishi
R
Dangaria
Jhanvi H
JH
Butler
Peter J
PJ
D001695:Biology / Q000295:instrumentation / Q000379:methods; D001696:Biomechanical Phenomena / Q000295:instrumentation* / Q000379:methods; D002469:Cell Separation / Q000295:instrumentation* / Q000379:methods; D004867:Equipment Design; D019544:Equipment Failure Analysis; D005434:Flow Cytometry / Q000295:instrumentation* / Q000379:methods; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation / Q000379:methods; D018613:Microscopy, Confocal / Q000295:instrumentation* / Q000379:methods; D008856:Microscopy, Fluorescence / Q000295:instrumentation* / Q000379:methods; D008967:Molecular Biology / Q000295:instrumentation / Q000379:methods; D052898:Optical Tweezers; D018511:Systems Integration