High Resolution, Large Deformation 3D Traction Force Microscopy

Abstract

The pyridine nucleotides (NADþ, NADPþ) are the major coenzymes participate in multiple redox processes in living cells. Both NADþ and NADPþ are not fluorescent and almost structurally identical, so it is difficult to directly distinguish NADþ or NADPþ via optical methods (such as fluorescence and Raman spectroscopy). We report here a sensitive probe of NADþ/NADPþ based on fluorescent silver nanoclusters with dual emission band. The silver nanoclusters with an initial fluorescent emission peak at 410 nm were synthesized by etching large size silver nanoparticles. With the addition of NADþ/ NADPþ solution, due to the strong coupling (charge-transfer) between silver nanoclusters and ligands (NADþ/NADPþ), a new fluorescence emission peak of the silver nanoclusters was found and raised at 550 nm and the fluorescence intensity was dependent on the ratio between NADþ and NADPþ. The time-resolved fluorescence decay (at 550 nm) of silver nanoclusters showed a single-exponential decay lifetime of 3.9 ns caused by the strong coupling between silver nanoclusters and ligands (NADþ/NADPþ). Meanwhile, the 410 nm emission was also selectively enhanced by the different ratio of NADþ /NADPþ molecules. The intensity ratio of fluorescence emission at 410nm and 550nm may be useful to monitor the levels of NADþ /NADPþ in aqueous solutions, cellular extracts and living cells. Candidate mechanisms and the analysis of time resolved emission spectra will be discussed.

Three-dimensional traction forces of Schwann cells on compliant substrates

“…Cell-generated full-field displacements and tractions were computed using our recently developed large deformation three-dimensional traction force microscopy (three-dimensional LDTFM) method [21]. Briefly, three-dimensional timelapsed volumetric images of fluorescent beads embedded in PA substrates were recorded using laser scanning confocal microscopy (LSCM).…”

“…The differentiation kernel we used in this study is an optimal-9 tap filter described in detail by Farid et al [23]. The computation of F is necessary to accurately calculate the Cauchy stress tensor, s, because for large deformations, as those seen in this study, the reference and deformed configurations are no longer identical [21]. For this reason, Hooke's law of linear small-strain elasticity used in most previous studies [24 -28] is replaced by a neo-Hookean finite deformation material model [29].…”

“…After removal of the excess water, 100 ml of sulfo-SANPAH (1 mg ml 21 ) was deposited onto the surface of each sample, and irradiated with ultraviolet light for 15 min to initiate activation. The darkened sulfo-SANPAH solution was aspirated, and the procedure was repeated.…”

“…Alexa 488-phalloidin methanolic stock stain solution was diluted 1:40 in PBS and added to samples for 1 h followed by two rinses with PBS. Cell nuclei were stained with TO-PRO-3 iodide (Sigma-Aldrich) diluted to 1 mg ml 21 in PBS. Samples were rinsed two times with PBS before they were imaged.…”

“…Using our recently improved three-dimensional large deformation traction force microscopy technique [21], we quantified the threedimensional SC displacement and traction field as a function of substrate stiffness. One of the key improvements in our technique is the incorporation of finite deformation constitutive models that allow proper determination of cell tractions in the presence of large deformations.…”