In nature, macroscopic excitation waves1,2 are found in a diverse range of settings including chemical reactions, metal rust, yeast, amoeba and the heart and brain. In the case of living biological tissue, the spatiotemporal patterns formed by these excitation waves are different in healthy and diseased states2,3. Current electrical and pharmacological methods for wave modulation lack the spatiotemporal precision needed to control these patterns. Optical methods have the potential to overcome these limitations, but to date have only been demonstrated in simple systems, such as the Belousov–Zhabotinsky chemical reaction4. Here, we combine dye-free optical imaging with optogenetic actuation to achieve dynamic control of cardiac excitation waves. Illumination with patterned light is demonstrated to optically control the direction, speed and spiral chirality of such waves in cardiac tissue. This all-optical approach offers a new experimental platform for the study and control of pattern formation in complex biological excitable systems.

Numerous dyes are available or under development for probing the structural and functional properties of biological membranes. Exogenous chromophores adopt a range of orientations when bound to membranes, which have a drastic effect on their biophysical behavior. Here, we present a method that employs optical anisotropy data from three polarization-imaging techniques to establish the distribution of orientations adopted by molecules in monolayers and bilayers. The resulting probability density functions, which contain the preferred molecular tilt μ and distribution breadth γ, are more informative than an average tilt angle [φ]. We describe a methodology for the extraction of anisotropy data through an image-processing technology that decreases the error in polarization measurements by about a factor of four. We use this technique to compare di-4-ANEPPS and di-8-ANEPPS, both dipolar dyes, using data from polarized 1-photon, 2-photon fluorescence and second-harmonic generation imaging. We find that di-8-ANEPPS has a lower tilt but the same distributional width. We find the distribution of tilts taken by di-4-ANEPPS in two phospholipid membrane models: giant unilamellar vesicles and water-in-oil droplet monolayers. Both models result in similar distribution functions with average tilts of 52° and 47°, respectively.

Objective: We developed a method for measuring SL and regional cell orientation using remote focusing microscopy, an emerging imaging modality that can capture light from arbitrary oblique planes within a sample. Methods and Results:We present a protocol that unambiguously and quickly determines cell orientation from user-selected areas in a field of view by imaging 2 oblique planes that share a common major axis with the cell. We demonstrate the effectiveness of the technique in establishing single-cell SL in Langendorff-perfused hearts loaded with the membrane dye di-4-ANEPPS. Conclusions:

Neurons communicate by using electrical signals, mediated by transient changes in the voltage across the plasma membrane. Optical techniques for visualizing these transmembrane potentials could revolutionize the field of neurobiology by allowing the spatial profile of electrical activity to be imaged in real time with high resolution, along individual neurons or groups of neurons within their native networks.1, 2 Second harmonic generation (SHG) is one of the most promising methods for imaging membrane potential, although so far this technique has only been demonstrated with a narrow range of dyes.3 Here we show that SHG from a porphyrin-based membrane probe gives a fast electro-optic response to an electric field which is about 5–10 times greater than that of conventional styryl dyes. Our results indicate that porphyrin dyes are promising probes for imaging membrane potential.