Integrated multimodal microscopy, time-resolved fluorescence, and optical-trap rheometry: toward single molecule mechanobiology

Abstract

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.

Fig. 1

Confocal volume for FCS. The radial dimension r of the confocal volume is close to the diffraction limit of the objective and is determined by keeping the (r/z) parameter constant (<10) in Eq. (6) when fitting the autocorrelation curve from R6G molecules in water. The radius thus obtained from experiment is 326 ± 10 nm. Fluorescent molecules are excited by the entire laser beam and fluorescence emission is only collected in the confocal volume. When particles move into and out of the confocal volume in the x, y, and z directions, 3-D diffusion is considered. When particles only move in the x-y plane, 2-D diffusion is considered.

Fig. 2

Optical setup. The Kr/Ar-ion, diode, or pulsed Nd:YAG laser beam is transmitted via fiber coupling to the TIRF or confocal port. For confocal illumination, upon exiting the fiber, the beam is collimated with lens L1, expanded by lenses L2 and L3, steered by the mirrors M1 and M2, reflected off the dichroic mirror (DM1), and enters the right side port of the microscope (note that the tube lens for the side port has been removed). After excitation of the sample, the fluorescence emission signal is collimated by the objective and exits the side port, passes through the dichroic mirror and is focused—using lens L4—onto the optical fiber which is connected to the photomultiplier tube (PMT). A polarizing beamsplitter (PBS) can be introduced before the fiber to separate light with polarization that is parallel or perpendicular to that of the excitation light. The PMTs convert single photons to electrical pulses, which are routed to the TCSPC board. Laser light from the TIRF system shares the back port of the microscope with the epifluorescence tube (Epi). Lenses L5 and L6 collimate the epifluorescence and TIRF light, respectively. The TIRF illumination is focused at the objective back aperture by the lens L7. When the sliding mirror, Mslide1, is removed from the light path, the right side port is closed and the emission signal can be collected by the camera via the tube lens (TL). In addition, the optical trap can be inserted above the fluorescence cube turret with an infrared dichroic mirror (DMslide) mounted on a custom-built slider (not shown).

Fig. 3

Custom micromanipulation chamber comprised of mounting plate, silicone spacers, metallic chamber with a side aperture, thermocouple for temperature monitoring, micropipette for micromanipulation, and the water circulation chamber.

Fig. 4

Sample autocorrelation curves for various concentrations of rhodamine 6G ranging from 1 to 10 nM; G(0) (inversely proportional to the average number of particles in the observation volume) was determined by fitting autocorrelation functions with Eq. (6). Inset: The average diffusion times τ_D of R6G molecules obtained from fitting autocorrelation curves with Eq. (6) (error bars indicate the standard deviation of 10 measurements).

Fig. 5

(a) Dependence of the autocorrelation curve of R6G on viscosity of glycerol/water solution, where an increase of viscosity (corresponding to increasing percentage of glycerol, v/v) leads to longer characteristic diffusion times τ_D, and (b) diffusion coefficients (D) of 5-nM R6G obtained by fitting autocorrelation curves in (a) with Eq. (6). The x axis indicates the values of viscosity of the dye solution measured with a cone-and-plate viscometer. Inset figure: The relationship between normalized viscosity (η/η₀) and normalized characteristic diffusion time (τ_D/τ_D0) fit with a line; η₀ and τ_D0 are viscosity and characteristic diffusion time, respectively, of dye in pure water.

Fig. 6

Bovine aortic endothelial cell imaged with DIC microscopy. Superimposed points in the image indicate location of confocal volume on the membrane surface of a representative cell from which fluorescence fluctuation data were collected. The cell in this figure corresponds to cell 1 in Table 2.

Fig. 7

(a) Representative plots of the autocorrelation of fluorescence intensity fluctuations arising from BAEC membranes stained with 5-nM DiI-C₁₈. Autocorrelation functions were fit with Eqs. (7)–(9) describing 2-D diffusion (one species), 2-D diffusion (two species), and anomalous diffusion, respectively. The residual of the 2-D, two-species fit is shown at the bottom of the graph (blue line). (b) Representative autocorrelation curve and 2-D diffusion fit of DiI-C₁₈ in a DMPC membrane. The residual of the fit is shown at the bottom of the graph.

Fig. 8

Fluorescence decay (blue line) and instrument response function (red line) for DiD fluorescence in ethanol. The decay histogram is fit with a double exponential (black line) to obtain the fluorescence lifetime. The χ² value of the fit is 1.17. The FWHM of the instrument response function is ~300 ps.

Fig. 9

TTL trap induced displacement of a 0.5-µm bead embedded in a 3% gelatin solution. The TTL trap can also operate in continuous mode. Trap stiffness was determined to be 11 pN/µm by tracking thermally induced displacements of trapped 0.5-µm polystyrene beads in DPBS and 1% albumin. The statistical variance of x coordinates for trapped beads was computed and compared to the potential energy and trap spring constant using Hooke’s law and the equipartition theorem. TTL modulation enables analysis of time-dependent responses of the surrounding environment of the bead in response to step changes in trap strength.

Fig. 10

DiI-C₁₈-stained bovine aortic endothelial cells imaged sequentially with DIC, epifluorescence, and TIRF.