PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When the substrate is coated with an extracellular matrix protein, cells adhere to the gel and apply forces, causing the gel to deform. The deformation depends on the cell traction and the elastic properties of the gel. If the deformation field of the surface is known, surface traction can be calculated using elasticity theory. Gel deformation is commonly measured by embedding fluorescent marker beads uniformly into the gel. The probes displace as the gel deforms. The probes near the surface of the gel are tracked. The displacements reported by these probes are considered as surface displacements. Their depths from the surface are ignored. This assumption introduces error in traction force evaluations. For precise measurement of cell forces, it is critical for the location of the beads to be known. We have developed a technique that utilizes simple chemistry to confine fluorescent marker beads, 0.1 and 1 µm in diameter, in PA gels, within 1.6 μm of the surface. We coat a coverslip with poly-D-lysine (PDL) and fluorescent beads. PA gel solution is then sandwiched between the coverslip and an adherent surface. The fluorescent beads transfer to the gel solution during curing. After polymerization, the PA gel contains fluorescent beads on a plane close to the gel surface. Video LinkThe video component of this article can be found at

The intracellular environment is a dynamic space filled with various organelles moving in all directions. Included in this diverse group of organelles are vesicles, which are involved in transport of molecular cargo throughout the cell. Vesicles move in either a directed or non-directed fashion, often depending on interactions with cytoskeletal proteins such as microtubules, actin filaments, and molecular motors. How these proteins affect the local fluctuations of vesicles in the cytoplasm is not clear since they have the potential to both facilitate and impede movement. Here we show that vesicle mobility is significantly affected by myosin-II, even though it is not a cargo transport motor. We find that myosin-II activity increases the effective diffusivity of vesicles and its inhibition facilitates longer states of non-directed motion. Our study suggests that altering myosin-II activity in the cytoplasm of cells can modulate the mobility of vesicles, providing a possible mechanism for cells to dynamically tune the cytoplasmic environment in space and time.

Illumination can have adverse effects on live cells. However, many experiments, e.g. traction force microscopy, rely on fluorescence microscopy. Current methods to assess undesired photo-induced cell changes rely on qualitative observation of changes in cell morphology. Here we utilize a quantitative technique to identify the effect of light on cell contractility prior to morphological changes. Fibroblasts were cultured on soft elastic hydrogels embedded with fluorescent beads. The adherent cells generated contractile forces that deform the substrate. Beads were used as fiducial markers to quantify the substrate deformation over time, which serves as a measure of cell force dynamics. We find that cells exposed to moderate fluorescence illumination (λ = 540–585 nm, I = 12.5 W/m2, duration = 60 s) exhibit rapid force relaxation. Strikingly, cells exhibit force relaxation after only 2 s of exposure, suggesting that photo-induced relaxation occurs nearly immediately. Evidence of photo-induced morphological changes were not observed for 15–30 min after illumination. Force relaxation and morphological changes were found to depend on wavelength and intensity of excitation light. This study demonstrates that changes in cell contractility reveal evidence of a photo-induced cell response long before any morphological cues.

Exposure to fluorescent excitation light is known to adversely affect living cells. Here, we provide experimental evidence showing fibroblast cells relax abruptly during illumination with fluorescent excitation light. Cells were plated on polyacrylamide (PA) gels embedded with fluorescent microbeads. The beads served as fiducial markers to track substrate displacements induced by cell force. When the cells adhere, they apply traction forces to their underlying surface, causing the substrate to contract inward with respect to the cell boundary. We find that contractile cell forces are sporadically interrupted by abrupt force relaxation events, characterized by outward "jumps" in displacement (≥ 30 nm in 0.5 s) of the underlying substrate during exposure to fluorescent light. Jumps occur more frequently on softer substrates. Additionally, the speed and direction of displacements of particles before and after jumps are conserved, suggesting that the jumps do not involve any change in cytoskeletal or adhesion structure of the cell, and are possibly mediated by disengagement and re-engagement of myosin motors with actin filaments. Interestingly, we find that the frequency of jumps is also modulated by the energy introduced by the excitation light source. We propose a mechanistic hypothesis to explain the observations.

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