Practical aspects of the cellular force inference toolkit (CellFIT).

If we are to fully understand the reasons that cells and tissues move and acquire their distinctive geometries during processes such as embryogenesis and wound healing, we will need detailed maps of the forces involved. One of the best current prospects for obtaining this information is noninvasive force-from-images techniques such as CellFIT, the Cellular Force Inference Toolkit, whose various steps are discussed here. Like other current quasistatic approaches, this one assumes that cell shapes are produced by interactions between interfacial tensions and intracellular pressures.

CellFIT: a cellular force-inference toolkit using curvilinear cell boundaries.

Mechanical forces play a key role in a wide range of biological processes, from embryogenesis to cancer metastasis, and there is considerable interest in the intuitive question, "Can cellular forces be inferred from cell shapes?" Although several groups have posited affirmative answers to this stimulating question, nagging issues remained regarding equation structure, solution uniqueness and noise sensitivity. Here we show that the mechanical and mathematical factors behind these issues can be resolved by using curved cell edges rather than straight ones. We present a new package of force-inference equations and assessment tools and denote this new package CellFIT, the Cellular Force Inference Toolkit.

Transmembrane current imaging in the heart during pacing and fibrillation.

Recently, we described a method to quantify the time course of total transmembrane current (Im) and the relative role of its two components, a capacitive current (Ic) and a resistive current (Iion), corresponding to the cardiac action potential during stable propagation. That approach involved recording high-fidelity (200 kHz) transmembrane potential (Vm) signals with glass microelectrodes at one site using a spatiotemporal coordinate transformation via measured conduction velocity. Here we extend our method to compute these transmembrane currents during stable and unstable propagation from fluorescence signals of Vm at thousands of sites (3 kHz), thereby introducing transmembrane current imaging.

Apical oscillations in amnioserosa cells: basolateral coupling and mechanical autonomy.

Holographic laser microsurgery is used to isolate single amnioserosa cells in vivo during early dorsal closure. During this stage of Drosophila embryogenesis, amnioserosa cells undergo oscillations in apical surface area. The postisolation behavior of individual cells depends on their preisolation phase in these contraction/expansion cycles: cells that were contracting tend to collapse quickly after isolation; cells that were expanding do not immediately collapse, but instead pause or even continue to expand for ∼40 s.

Quantification of transmembrane currents during action potential propagation in the heart.

The measurement, quantitative analysis, theory, and mathematical modeling of transmembrane potential and currents have been an integral part of the field of electrophysiology since its inception. Biophysical modeling of action potential propagation begins with detailed ionic current models for a patch of membrane within a distributed cable model. Voltage-clamp techniques have revolutionized clinical electrophysiology via the characterization of the transmembrane current gating variables; however, this kinetic information alone is insufficient to accurately represent propagation.

Enabling user-guided segmentation and tracking of surface-labeled cells in time-lapse image sets of living tissues.

To study the process of morphogenesis, one often needs to collect and segment time-lapse images of living tissues to accurately track changing cellular morphology. This task typically involves segmenting and tracking tens to hundreds of individual cells over hundreds of image frames, a scale that would certainly benefit from automated routines; however, any automated routine would need to reliably handle a large number of sporadic, and yet typical problems (e.g.

A phased-array stimulator system for studying planar and curved cardiac activation wavefronts.

Wavefront propagation in cardiac tissue is affected greatly by the geometry of the wavefront. We describe a computer-controlled stimulator system that creates reproducible wavefronts of a predetermined shape and orientation for the investigation of the effects of wavefront geometry. We conducted demonstration experiments on isolated perfused rabbit hearts, which were stained with the voltage-sensitive dye, di-4-ANEPPS.

A high-voltage cardiac stimulator for field shocks of a whole heart in a bath.

Defibrillators are a critical tool for treating heart disease; however, the mechanisms by which they halt fibrillation are still not fully understood and are the subject of ongoing research. Clinical defibrillators do not provide the precise control of shock timing, duration, and voltage or other features needed for detailed scientific inquiry, and there are few, if any, commercially available units designed for research applications. For this reason, we have developed a high-voltage, programmable, capacitive-discharge stimulator optimized to deliver defibrillation shocks with precise timing and voltage control to an isolated animal heart, either in air or in a bath.