Recent advances in optogenetics have opened new routes to drug discovery, particularly in neuroscience. Physiological cellular assays probe functional phenotypes that connect genomic data to patient health. Optogenetic tools, in particular tools for all-optical electrophysiology, now provide a means to probe cellular disease models with unprecedented throughput and information content. These techniques promise to identify functional phenotypes associated with disease states and to identify compounds that improve cellular function regardless of whether the compound acts directly on a target or through a bypass mechanism. This review discusses opportunities and unresolved challenges in applying optogenetic techniques throughout the discovery pipeline – from target identification and validation, to target-based and phenotypic screens, to clinical trials.

•Recent advances in genetically encoded voltage indicators (GEVIs) have brought neural voltage imaging in vivo within reach.•Recently introduced tools such as electrically spiking HEK cells facilitate rapid testing of new GEVIs.•Light-gated voltage integrators and reporters of absolute voltage reporters implement new forms of molecular logic.•Many potentially useful GEVI scaffolds and designs remain to be tested.•Improvements in microscopy and software will be needed to attain full benefit from the newest GEVIs.Membrane voltages are ubiquitous throughout cell biology. Voltage is most commonly associated with excitable cells such as neurons and cardiomyocytes, although many other cell types and organelles also support electrical signaling. Voltage imaging in vivo would offer unique capabilities in reporting the spatial pattern and temporal dynamics of electrical signaling at the cellular and circuit levels. Voltage is not directly visible, and so a longstanding challenge has been to develop genetically encoded fluorescent voltage indicator proteins. Recent advances have led to a profusion of new voltage indicators, based on different scaffolds and with different tradeoffs between voltage sensitivity, speed, brightness, and spectrum. In this review, we describe recent advances in design and applications of genetically-encoded voltage indicators (GEVIs). We also highlight the protein engineering strategies employed to improve the dynamic range and kinetics of GEVIs and opportunities for future advances.Download high-res image (180KB)Download full-size image

Photons are a fascinating reagent, flowing and reacting quite differently compared to more massive and less ephemeral particles of matter. The optogenetic palette comprises an ever growing set of light-responsive proteins, which open the possibility of using light to perturb and to measure biological processes with great precision in space and time. Yet there are limits on what light can achieve. Diffraction limits the smallest features, and scattering in tissue limits the largest. Photobleaching, diffusion of photogenerated products, and optical crosstalk between overlapping absorption spectra further muddy the optogenetic picture, particularly when one wants to use multiple optogenetic tools simultaneously.But these obstacles are surmountable. Most light-responsive proteins and small molecules undergo more than one light-driven transition, often with different action spectra and kinetics. By overlapping multiple laser beams, carefully patterned in space, time, and wavelength, one can steer molecules into fluorescent or nonfluorescent, active or inactive conformations. By doing so, one can often circumvent the limitations of simple one-photon excitation and achieve new imaging and stimulation capabilities. These include subdiffraction spatial resolution, optical sectioning, robustness to light scattering, and multiplexing of more channels than can be achieved with simple one-photon excitation.The microbial rhodopsins are a particularly rich substrate for this type of multiphoton optical control. The natural diversity of these proteins presents a huge range of starting materials. The spectroscopy and photocycles of microbial rhodopsins are relatively well understood, providing states with absorption maxima across the visible spectrum, which can be accessed on experimentally convenient time scales. A long history of mutational studies in microbial rhodopsins allows semirational protein engineering. Mutants of Archaerhodopsin 3 (Arch) come in all the colors of the rainbow. In a solution of purified Arch-eGFP, a focused green laser excites eGFP fluorescence throughout the laser path, while a focused red laser excites fluorescence of Arch only near the focus, indicative of multiphoton fluorescence. This nonlinearity occurs at a laser intensity ∼1010-fold lower than in conventional two-photon microscopy! The mutant Arch(D95H) shows photoswitchable optical bistability. In a lawn of E. coli expressing this mutant, illumination with patterned blue light converts the molecule into a state that is fluorescent. Illumination with red light excites this fluorescence, and gradually resets the molecules back to the non-fluorescent state. This review describes the new types of molecular logic that can be implemented with multi-photon control of microbial rhodopsins, from whole-brain activity mapping to measurements of absolute membrane voltage.Part of our goal in this Account is to describe recent work in nonlinear optogenetics, but we also present a variety of interesting things one could do if only the right optogenetic molecules were available. This latter component is intended to inspire future spectroscopic, protein discovery, and protein engineering work.

Sorting of target cells from a heterogeneous pool is technically difficult when the selection criterion is complex, e.g. a dynamic response, a morphological feature, or a combination of multiple parameters. At present, mammalian cell selections are typically performed either via static fluorescence (e.g. fluorescence activated cell sorter), via survival (e.g. antibiotic resistance), or via serial operations (flow cytometry, laser capture microdissection). Here we present a simple protocol for selecting cells based on any static or dynamic property that can be identified by video microscopy and image processing. The “photostick” technique uses a cell-impermeant photochemical crosslinker and digital micromirror array-based patterned illumination to immobilize selected cells on the culture dish. Other cells are washed away with mild protease treatment. The crosslinker also labels the selected cells with a fluorescent dye and a biotin for later identification. The photostick protocol preserves cell viability, permits genetic profiling of selected cells, and can be performed with complex functional selection criteria such as neuronal firing patterns.

We developed a technique, “flash memory”, to record a photochemical imprint of the activity state—firing or not firing—of a neuron at a user-selected moment in time. The key element is an engineered microbial rhodopsin protein with three states. Two nonfluorescent states, D1 and D2, exist in a voltage-dependent equilibrium. A stable fluorescent state, F, is reached by a photochemical conversion from D2. When exposed to light of a wavelength λwrite, population transfers from D2 to F, at a rate determined by the D1 ⇌ D2 equilibrium. The population of F maintains a record of membrane voltage which persists in the dark. Illumination at a later time at a wavelength λread excites fluorescence of F, probing this record. An optional third flash at a wavelength λreset converts F back to D2, for a subsequent write–read cycle. The flash memory method offers the promise to decouple the recording of neural activity from its readout. In principle, the technique may enable one to generate snapshots of neural activity in a large volume of neural tissue, e.g., a complete mouse brain, by circumventing the challenge of imaging a large volume with simultaneous high spatial and high temporal resolution. The proof-of-principle flash memory sensors presented here will need improvements in sensitivity, speed, brightness, and membrane trafficking before this goal can be realized.

We show that the solid–solid friction between bulk chiral molecular solids can depend on the relative chirality of the two materials. In menthol and 1-phenyl-1-butanol, heterochiral friction is smaller than homochiral friction, while in ibuprofen, heterochiral friction is larger. Chiral asymmetries in the coefficient of sliding friction vary with temperature and can be as large as 30%. In the three compounds tested, the sign of the difference between heterochiral and homochiral friction correlated with the sign of the difference in melting point between racemate (compound or conglomerate) and pure enantiomer. Menthol and ibuprofen each form a stable racemic compound, while 1-phenyl-1-butanol forms a racemic conglomerate. Thus, a difference between heterochiral and homochiral friction does not require the formation of a stable interfacial racemic compound. Measurements of chirality-dependent friction provide a unique means to distinguish the role of short-range intermolecular forces from all other sources of dissipation in the friction of bulk molecular solids.

Microbial rhodopsins were recently introduced as genetically encoded fluorescent indicators of membrane voltage. An understanding of the mechanism underlying this function would aid in the design of improved voltage indicators. We asked, what states can the protein adopt, and which states are fluorescent? How does membrane voltage affect the photostationary distribution of states? Here, we present a detailed spectroscopic characterization of Archaerhodopsin 3 (Arch). We performed fluorescence spectroscopy on Arch and its photogenerated intermediates in Escherichia coli and in single HEK293 cells under voltage-clamp conditions. These experiments probed the effects of time-dependent illumination and membrane voltage on absorption, fluorescence, membrane current, and membrane capacitance. The fluorescence of Arch arises through a sequential three-photon process. Membrane voltage modulates protonation of the Schiff base in a 13-cis photocycle intermediate (M ⇌ N equilibrium), not in the ground state as previously hypothesized. We present experimental protocols for optimized voltage imaging with Arch, and we discuss strategies for engineering improved rhodopsin-based voltage indicators.

We developed a system to reversibly encapsulate small numbers of molecules in an array of nanofabricated “dimples”. This system enables highly parallel, long-term, and attachment-free studies of molecular dynamics via single-molecule fluorescence. In studies of bimolecular reactions of small numbers of confined molecules, we see phenomena that, while expected from basic statistical mechanics, are not observed in bulk chemistry. Statistical fluctuations in the occupancy of sealed reaction chambers lead to steady-state fluctuations in reaction equilibria and rates. These phenomena are likely to be important whenever reactions happen in confined geometries.

Biological hydrogels are continuously turned over through secretion and degradation. This non-equilibrium flux may be important for cellular and molecular transport through mucus and the extracellular matrix. Gel-digesting enzymes can drastically change the physical and chemical properties of the hydrogel environment. We report that a spatial gradient in the degradation of two gel/enzyme systems—gelatin/trypsin and hyaluronan/hyaluronidase—leads to directional motion of particles embedded in the gel in the direction of higher enzyme concentration. We study the rate at which the degradation front propagates through the gel and the ensuing velocity of the embedded particles, as functions of enzyme and gel concentrations. We propose that asymmetric degradation leads to asymmetric swelling, which transports particles up the enzyme concentration gradient.

We used a fluorescent chemical indicator of magnetic field to visualize the magnetic field around ferromagnetic nanostructures. The indicator was a chain-linked electron donor–acceptor molecule, phenanthrene-(CH2)12-O-(CH2)2-dimethylaniline, that forms spin-correlated radical pairs upon photoexcitation. The magnetic field altered the coherent spin dynamics, yielding an 80% increase in exciplex fluorescence in a 0.1 T magnetic field. The magnetic field distributions were quantified to precision of 1.8 × 10–4 T by image analysis and agreed with finite-element nanomagnetic simulations.

Anti-Brownian electrokinetic traps have been used to trap and study the free-solution dynamics of large protein complexes and long chains of DNA. Small molecules in solution have thus far proved too mobile to trap by any means. Here we explore the ultimate limits on trapping single molecules. We developed a feedback-based anti-Brownian electrokinetic trap in which classical thermal noise is compensated to the maximal extent allowed by quantum measurement noise. We trapped single fluorophores with a molecular weight of < 1 kDa and a hydrodynamic radius of 6.7 Å for longer than one second, in aqueous buffer at room temperature. This achievement represents an 800-fold decrease in the mass of objects trapped in solution, and opens the possibility to trap and manipulate any soluble molecule that can be fluorescently labeled. To illustrate the use of this trap, we studied the binding of unlabeled RecA to fluorescently labeled single-stranded DNA. Binding of RecA induced changes in the DNA diffusion coefficient, electrophoretic mobility, and brightness, all of which were measured simultaneously and on a molecule-by-molecule basis. This device greatly extends the size range of molecules that can be studied by room temperature feedback trapping, and opens the door to further studies of the binding of unmodified proteins to DNA in free solution.

It has recently become possible to trap individual fluorescent biomolecules in aqueous solution by using real-time tracking and active feedback to suppress Brownian motion. We propose areas of investigation in which anti-Brownian electrokinetic (ABEL) trapping of single molecules is likely to lead to significant new insights into biomolecular dynamics.

Electromagnetic fields with complex spatial variation routinely arise in Nature. We study the response of a small molecule to monochromatic fields of arbitrary three-dimensional geometry. First, we consider the allowed configurations of the fields and field gradients at a single point in space. Many configurations cannot be generated from a single plane wave, regardless of polarization, but any allowed configuration can be generated by superposition of multiple plane waves. There is no local configuration of the fields and gradients that requires near-field effects. Second, we derive a set of local electromagnetic quantities, each of which couples to a particular multipole transition. These quantities are small or zero in plane waves, but can be large in regions of certain superpositions of plane waves. Our findings provide a systematic framework for designing far-field and near-field experiments to drive multipole transitions. The proposed experiments provide information on molecular structure that is inaccessible to other spectroscopic techniques and open the possibility for new types of optical control of molecules.

Fluorescence imaging is used to study the dynamics of a wide variety of single molecules in solution or attached to a surface. Two key challenges in this pursuit are (1) to image immobilized single molecules in the presence of a high level of fluorescent background and (2) to image freely diffusing single molecules for long times. Strategies that perform well by one measure often perform poorly by the other. Here, we present a simple modification to a wide-field fluorescence microscope that addresses both challenges and dramatically improves single-molecule imaging. The technique of convex lens-induced confinement (CLIC) restricts molecules to a wedge-shaped gap of nanoscale depth, formed between a plano-convex lens and a planar coverslip. The shallow depth of the imaging volume leads to 20-fold greater rejection of background fluorescence than is achieved with total internal reflection fluorescence (TIRF) imaging. Elimination of out-of-plane diffusion leads to an approximately 10 000-fold longer diffusion-limited observation time per molecule than is achieved with confocal fluorescence correlation spectroscopy. The CLIC system also provides a new means to determine molecular size. The CLIC system does not require any nanofabrication, nor any custom optics, electronics, or computer control.

We report enhanced optical Faraday rotation in gold-coated maghemite (γ-Fe2O3) nanoparticles. The Faraday rotation spectrum measured from 480−690 nm shows a peak at about 530 nm, not present in either uncoated maghemite nanoparticles or solid gold nanoparticles. This peak corresponds to an intrinsic electronic transition in the maghemite nanoparticles and is consistent with a near-field enhancement of Faraday rotation resulting from the spectral overlap of the surface plasmon resonance in the gold with the electronic transition in maghemite. This demonstration of surface plasmon resonance-enhanced magneto-optics (SuPREMO) in a composite magnetic/plasmonic nanosystem may enable design of nanostructures for remote sensing and imaging of magnetic fields and for miniaturized magneto-optical devices.

Metallic and magnetic nanostructures set electromagnetic boundary conditions which can lead to highly contorted fields in their immediate vicinity. While much attention has been devoted to enhancements in electric field strength, we argue that equally interesting phenomena arise from enhancements in magnetic and electric field gradients. Nonuniform fields near nanostructures can induce molecular transitions that are forbidden by electric dipole selection rules. We illustrate this claim with two examples. “Superhelical” electromagnetic fields are predicted to show enhanced asymmetry in their interaction with chiral molecules, far greater than that due to circularly polarized light. Such fields could be used to induce chiral photochemistry with large enantiomeric excess. Steeply varying DC magnetic fields are predicted to enhance the rate of intersystem crossing in molecular bi-radicals. Such fields could provide a route to new nanomagnetic catalysts and to magnetic control of chemical reactions.

Fluorescent imaging of single helicene molecules is applied to study the optical activity of chiral fluorophores. In contrast to the previous report by Hassey et al. (Science 2006, 314, 1437), the dissymmetry factors of single chiral fluorophores are found not to differ significantly from the bulk value of |g| < 10−4 at 457 nm. Linear dichroism and birefringence of the dichroic mirror inside the fluorescence microscope change the polarization state of the incoming laser beam significantly; i.e., circular polarized light sent into the microscope becomes highly elliptically polarized after reflection from the dichroic mirror. Compensation for this effect should be made to avoid artifacts brought by linear dichroism in single immobilized molecules.

To study the impact of neural activity on cellular physiology, one would like to combine precise control of firing patterns with highly sensitive probes of cellular physiology. Light-gated ion channels, e.g., Channelrhodopsin-2, enable precise control of firing patterns; green fluorescent protein-based reporters, e.g., the GCaMP6f Ca2+ reporter, enable highly sensitive probing of cellular physiology. However, for most actuator-reporter combinations, spectral overlap prevents straightforward combination within a single cell. Here we explore multiwavelength control of channelrhodopsins to circumvent this limitation. The “stoplight” technique described in this article uses channelrhodopsin variants that are opened by blue light and closed by orange light. Cells are illuminated with constant blue light to excite fluorescence of a green fluorescent protein-based reporter. Modulated illumination with orange light negatively regulates activation of the channelrhodopsin. We performed detailed photophysical characterization and kinetic modeling of four candidate stoplight channelrhodopsins. The variant with the highest contrast, sdChR(C138S,E154A), enabled all-optical measurements of activity-induced calcium transients in cultured rat hippocampal neurons, although cell-to-cell variation in expression levels presents a challenge for quantification.

Genetically encoded voltage indicators (GEVIs) can report cellular electrophysiology with high resolution in space and time. Two-photon (2P) fluorescence has been explored as a means to image voltage in tissue. Here, we used the 2P electronic excited-state lifetime to probe absolute membrane voltage in a manner that is insensitive to the protein expression level, illumination intensity, or photon detection efficiency. First, we tested several GEVIs for 2P brightness, response speed, and voltage sensitivity. ASAP1 and a previously described citrine-Arch electrochromic Förster resonance energy transfer sensor (dubbed CAESR) showed the best characteristics. We then characterized the voltage-dependent lifetime of ASAP1, CAESR, and ArcLight under voltage-clamp conditions. ASAP1 and CAESR showed voltage-dependent lifetimes, whereas ArcLight did not. These results establish 2P fluorescence lifetime imaging as a viable means of measuring absolute membrane voltage. We discuss the prospects and improvements necessary for applications in tissue.

Sorting of target cells from a heterogeneous pool is technically difficult when the selection criterion is complex, e.g. a dynamic response, a morphological feature, or a combination of multiple parameters. At present, mammalian cell selections are typically performed either via static fluorescence (e.g. fluorescence activated cell sorter), via survival (e.g. antibiotic resistance), or via serial operations (flow cytometry, laser capture microdissection). Here we present a simple protocol for selecting cells based on any static or dynamic property that can be identified by video microscopy and image processing. The “photostick” technique uses a cell-impermeant photochemical crosslinker and digital micromirror array-based patterned illumination to immobilize selected cells on the culture dish. Other cells are washed away with mild protease treatment. The crosslinker also labels the selected cells with a fluorescent dye and a biotin for later identification. The photostick protocol preserves cell viability, permits genetic profiling of selected cells, and can be performed with complex functional selection criteria such as neuronal firing patterns.