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We describe the design, construction, and application of what are believed to be the smallest fiber-optic probes used to date during imaging or diagnosis involving low-coherence interferometry (LCI). The probes use novel fiber-optic gradient-index (GRIN) lenses fabricated by a recently developed modified chemical-vapor-deposition (MCVD) process that avoids on-axis aberrations commonly marring MCVD-fabricated GRIN substrate. Fusing GRIN fiber lenses onto single-mode fiber yields automatically aligned all-fiber probes that insert into tissue through hypodermic needles as small as 31-gauge (inner diameter, 127 mum). We demonstrate the use of such probes with LCI by measuring microscopic brain motions in vivo.

A basic question for theories of learning and memory is whether neuronal plasticity suffices to guide proper memory recall. Alternatively, information processing that is additional to readout of stored memories might occur during recall. We formulate a “lock-and-key” hypothesis regarding cerebellum-dependent motor memory in which successful learning shapes neural activity to match a temporal filter that prevents expression of stored but inappropriate motor responses. Thus, neuronal plasticity by itself is necessary but not sufficient to modify motor behavior. We explored this idea through computational studies of two cerebellar behaviors and examined whether deep cerebellar and vestibular nuclei neurons can filter signals from Purkinje cells that would otherwise drive inappropriate motor responses. In eyeblink conditioning, reflex acquisition requires the conditioned stimulus (CS) to precede the unconditioned stimulus (US) by >100 ms. In our biophysical models of cerebellar nuclei neurons this requirement arises through the phenomenon of postinhibitory rebound depolarization and matches longstanding behavioral data on conditioned reflex timing and reliability. Although CS–US intervals 100 ms. This bound reflects the minimum time for deinactivation of rebound currents such as T-type Ca2+. In vestibulo-ocular reflex adaptation, hyperpolarization-activated currents in vestibular nuclei neurons may underlie analogous dependence of adaptation magnitude on the timing of visual and vestibular stimuli. Thus, the proposed lock-and-key mechanisms link channel kinetics to recall performance and yield specific predictions of how perturbations to rebound depolarization affect motor expression.

Towards overcoming the size limitations of conventional two-photon fluorescence microscopy for brain imaging in freely moving mice, we introduce a portable laser-scanning microendoscope based on a microelectromechanical systems (MEMS) two-dimensional (2-D) scanning mirror, compound gradient refractive index (GRIN) micro-lenses, and a photonic bandgap fiber (PBF). The microendoscope achieves fast line scanning acquisition rates up to 3.5 kHz and micron-scale imaging resolution.

The combination of micro- and fiber-optics enables minimally invasive nonlinear optical imaging in live subjects by microendoscopy. I will describe the develpment and application of two-photon excited fluorescence and second-harmonic generation microendoscopy in the mammalian nervous system. Article not available.

We sought to develop techniques for visualizing cochlear blood flow in live mammalian subjects using fluorescence microendoscopy.Inner ear microcirculation appears to be intimately involved in cochlear function. Blood velocity measurements suggest that intense sounds can alter cochlear blood flow. Disruption of cochlear blood flow may be a significant cause of hearing impairment, including sudden sensorineural hearing loss. However, inability to image cochlear blood flow in a nondestructive manner has limited investigation of the role of inner ear microcirculation in hearing function. Present techniques for imaging cochlear microcirculation using intravital light microscopy involve extensive perturbations to cochlear structure, precluding application in human patients. The few previous endoscopy studies of the cochlea have suffered from optical resolution insufficient for visualizing cochlear microvasculature. Fluorescence microendoscopy is an emerging minimally invasive imaging modality that provides micron-scale resolution in tissues inaccessible to light microscopy. In this article, we describe the use of fluorescence microendoscopy in live guinea pigs to image capillary blood flow and movements of individual red blood cells within the basal turn of the cochlea.We anesthetized eight adult guinea pigs and accessed the inner ear through the mastoid bulla. After intravenous injection of fluorescein dye, we made a limited cochleostomy and introduced a compound doublet gradient refractive index endoscope probe 1 mm in diameter into the inner ear. We then imaged cochlear blood flow within individual vessels in an epifluorescence configuration using one-photon fluorescence microendoscopy.We observed single red blood cells passing through individual capillaries in several cochlear structures, including the round window membrane, spiral ligament, osseous spiral lamina, and basilar membrane. Blood flow velocities within inner ear capillaries varied widely, with observed speeds reaching up to approximately 500 microm/s.Fluorescence microendoscopy permits visualization of cochlear microcirculation with micron-scale optical resolution and determination of blood flow velocities through analysis of video sequences.

Towards overcoming the size limitations of conventional two-photon fluorescence microscopy, we introduce laser-scanning instrumentation based on a microelectromechanical systems (MEMS) scanner and describe two-photon microscopy and microendoscopy with fast-axis acquisition rates up to 3.5 kHz. (180.2520) Fluorescence microscopy (180.5810) Scanning microscopy

Optical fibers guide light between separate locations and enable new types of fluorescence imaging. Fiber-optic fluorescence imaging systems include portable handheld microscopes, flexible endoscopes well suited for imaging within hollow tissue cavities and microendoscopes that allow minimally invasive high-resolution imaging deep within tissue. A challenge in the creation of such devices is the design and integration of miniaturized optical and mechanical components. Until recently, fiber-based fluorescence imaging was mainly limited to epifluorescence and scanning confocal modalities. Two new classes of photonic crystal fiber facilitate ultrashort pulse delivery for fiber-optic two-photon fluorescence imaging. An upcoming generation of fluorescence imaging devices will be based on microfabricated device components.

We introduce a compact two-photon fluorescence microendoscope based on a compound gradient refractive index endoscope probe, a DC micromotor for remote adjustment of the image plane, and a flexible photonic bandgap fiber for near distortion-free delivery of ultrashort excitation pulses. The imaging head has a mass of only 3.9?g and provides micrometer-scale resolution. We used portable two-photon microendoscopy to visualize hippocampal blood vessels in the brains of live mice.

Visual information reaches the brain by way of a fine cable, the optic nerve. The million or so axons in the optic nerve represent an information bottleneck in the visual pathway-where the fewest number of neurons convey the visual scene. It has long been thought that to make the most of the optic nerve's limited capacity the retina may encode visual information in an optimally efficient manner. In this issue of Neuron, Puchalla et al. report a test of this hypothesis using multielectrode recordings from retinal ganglion cells stimulated with movies of natural scenes. The authors find substantial redundancy in the retinal code and estimate that there is an approximately 10-fold overrepresentation of visual information.

We introduce a compact and lightweight (3.7 g) two-photon fluorescence microendoscope, which is based on a flexible photonic bandgap fiber and a DC micromotor, and which is designed for brain imaging in freely moving mice.

Innovations in two-photon fluorescence microscopy are extending the range of scientific issues and biological tissues that can be studied with in vivo cellular level imaging. Previously inacessible deep tissues can now be studied using two-photon fluorescence microendoscopy, fiber optic studies in behaving animals are under way, and new fluorescence staining methods are bringing in vivo cellular dynamics into view. Full-text article is not available.

Natural selection has created many species in which individual survival rests on computations performed by the organism's own physiology.

Imaging biological processes in mammalian tissues will be facilitated by fluorescent probes with excitation and emission bands within the near-infrared optical window of high transparency. Here we report a phytochrome-based near-infrared fluorescent protein (iRFP) with excitation and emission maxima at 690 nm and 713 nm, respectively. iRFP does not require an exogenous supply of the chromophore biliverdin and has higher effective brightness, intracellular stability and photostability than earlier phytochrome-derived fluorescent probes. Compared with far-red GFP-like proteins, iRFP has a substantially higher signal-to-background ratio in a mouse model due to its infrared-shifted spectra.

Therapeutic drugs for cognitive and psychiatric disorders are often characterized by their molecular mechanism of action. Here we demonstrate a new approach to elucidate drug action on large-scale neuronal activity by tracking somatic calcium dynamics in hundreds of CA1 hippocampal neurons of pharmacologically manipulated behaving mice. We used an adeno-associated viral vector to express the calcium sensor GCaMP3 in CA1 pyramidal cells under control of the CaMKII promoter and a miniaturized microscope to observe cellular dynamics. We visualized these dynamics with and without a systemic administration of Zolpidem, a GABAA agonist that is the most commonly prescribed drug for the treatment of insomnia in the United States. Despite growing concerns about the potential adverse effects of Zolpidem on memory and cognition, it remained unclear whether Zolpidem alters neuronal activity in the hippocampus, a brain area critical for cognition and memory. Zolpidem, when delivered at a dose known to induce and prolong sleep, strongly suppressed CA1 calcium signaling. The rate of calcium transients after Zolpidem administration was significantly lower compared to vehicle treatment. To factor out the contribution of changes in locomotor or physiological conditions following Zolpidem treatment, we compared the cellular activity across comparable epochs matched by locomotor and physiological assessments. This analysis revealed significantly depressive effects of Zolpidem regardless of the animal's state. Individual hippocampal CA1 pyramidal cells differed in their responses to Zolpidem with the majority (∼ 65%) significantly decreasing the rate of calcium transients, and a small subset (3%) showing an unexpected and significant increase. By linking molecular mechanisms with the dynamics of neural circuitry and behavioral states, this approach has the potential to contribute substantially to the development of new therapeutics for the treatment of CNS disorders.

Genetically encoded fluorescence voltage sensors offer the possibility of directly visualizing neural spiking dynamics in cells targeted by their genetic class or connectivity. Sensors of this class have generally suffered performance-limiting tradeoffs between modest brightness, sluggish kinetics and limited signalling dynamic range in response to action potentials. Here we describe sensors that use fluorescence resonance energy transfer (FRET) to combine the rapid kinetics and substantial voltage-dependence of rhodopsin family voltage-sensing domains with the brightness of genetically engineered protein fluorophores. These FRET-opsin sensors significantly improve upon the spike detection fidelity offered by the genetically encoded voltage sensor, Arclight, while offering faster kinetics and higher brightness. Using FRET-opsin sensors we imaged neural spiking and sub-threshold membrane voltage dynamics in cultured neurons and in pyramidal cells within neocortical tissue slices. In live mice, rates and optical waveforms of cerebellar Purkinje neurons' dendritic voltage transients matched expectations for these cells' dendritic spikes.

Motor proteins such as kinesin, myosin and polymerase convert chemical energy into work through a cycle that involves nucleotide hydrolysis. Kinetic rates in the cycle that depend upon load identify transitions at which structural changes, such as power strokes or diffusive motions, are likely to occur. Here we show, by modelling data obtained with a molecular force clamp, that kinesin mechanochemistry can be characterized by a mechanism in which a load-dependent isomerization follows ATP binding. This model quantitatively accounts for velocity data over a wide range of loads and ATP levels, and indicates that movement may be accomplished through two sequential 4-nm substeps. Similar considerations account for kinesin processivity, which is found to obey a load-dependent Michaelis-Menten relationship.

Micro-optics are increasingly used for minimally invasive in vivo imaging, in miniaturized microscopes and in lab-on-a-chip devices. Owing to optical aberrations and lower numerical apertures, a main class of microlens, gradient refractive index lenses, has not achieved resolution comparable to conventional microscopy. Here we describe high-resolution microlenses, and illustrate two-photon imaging of dendritic spines on hippocampal neurons and dual-color nonlinear optical imaging of neuromuscular junctions in live mice.

Kinesin is a two-headed, ATP-driven motor protein that moves processively along microtubules in discrete steps of 8 nm, probably by advancing each of its heads alternately in sequence. Molecular details of how the chemical energy stored in ATP is coupled to mechanical displacement remain obscure. To shed light on this question, a force clamp was constructed, based on a feedback-driven optical trap capable of maintaining constant loads on single kinesin motors. The instrument provides unprecedented resolution of molecular motion and permits mechanochemical studies under controlled external loads. Analysis of records of kinesin motion under variable ATP concentrations and loads revealed several new features. First, kinesin stepping appears to be tightly coupled to ATP hydrolysis over a wide range of forces, with a single hydrolysis per 8-nm mechanical advance. Second, the kinesin stall force depends on the ATP concentration. Third, increased loads reduce the maximum velocity as expected, but also raise the apparent Michaelis-Menten constant. The kinesin cycle therefore contains at least one load-dependent transition affecting the rate at which ATP molecules bind and subsequently commit to hydrolysis. It is likely that at least one other load-dependent rate exists, affecting turnover number. Together, these findings will necessitate revisions to our understanding of how kinesin motors function.

RNA polymerase (RNAP) moves along DNA while carrying out transcription, acting as a molecular motor. Transcriptional velocities for single molecules of Escherichia coli RNAP were measured as progressively larger forces were applied by a feedback-controlled optical trap. The shapes of RNAP force-velocity curves are distinct from those of the motor enzymes myosin or kinesin, and indicate that biochemical steps limiting transcription rates at low loads do not generate movement. Modeling the data suggests that high loads may halt RNAP by promoting a structural change which moves all or part of the enzyme backwards through a comparatively large distance, corresponding to 5 to 10 base pairs. This contrasts with previous models that assumed force acts directly upon a single-base translocation step.

A longstanding goal in neuroscience has been to develop techniques for imaging the voltage dynamics of genetically defined subsets of neurons. Optical sensors of transmembrane voltage would enhance studies of neural activity in contexts ranging from individual neurons cultured in vitro to neuronal populations in awake-behaving animals. Recent progress has identified Archaerhodopsin (Arch) based sensors as a promising, genetically encoded class of fluorescent voltage indicators that can report single action potentials. Wild-type Arch exhibits sub-millisecond fluorescence responses to trans-membrane voltage, but its light-activated proton pump also responds to the imaging illumination. An Arch mutant (Arch-D95N) exhibits no photocurrent, but has a slower, ~40 ms response to voltage transients. Here we present Arch-derived voltage sensors with trafficking signals that enhance their localization to the neural membrane. We also describe Arch mutant sensors (Arch-EEN and -EEQ) that exhibit faster kinetics and greater fluorescence dynamic range than Arch-D95N, and no photocurrent at the illumination intensities normally used for imaging. We benchmarked these voltage sensors regarding their spike detection fidelity by using a signal detection theoretic framework that takes into account the experimentally measured photon shot noise and optical waveforms for single action potentials. This analysis revealed that by combining the sequence mutations and enhanced trafficking sequences, the new sensors improved the fidelity of spike detection by nearly three-fold in comparison to Arch-D95N.

Although in vivo microscopy has been pivotal in enabling studies of neuronal structure and function in the intact mammalian brain, conventional intravital microscopy has generally been limited to superficial brain areas such as the olfactory bulb, the neocortex, or the cerebellar cortex. For imaging cells in deeper areas, this article discusses in vivo optical microendoscopy using gradient refractive index (GRIN) microlenses that can be inserted into tissue. Our general methodology is broadly applicable to many deep brain regions and areas of the body. Microendoscopes are available in a wide variety of optical designs, allowing imaging across a range of spatial scales and with spatial resolution that can now closely approach that offered by standard water-immersion microscope objectives. The incorporation of microendoscope probes into portable miniaturized microscopes allows imaging in freely behaving animals. When combined with the broad sets of available fluorescent markers, animal preparations, and genetically modified mice, microendoscopic methods enable sophisticated experimental designs for probing how cellular characteristics may underlie or reflect animal behavior and life experience, in healthy animals and animal models of disease.

Conventional intravital microscopy has generally been limited to superficial brain areas such as the olfactory bulb, the neocortex, or the cerebellar cortex. In vivo optical microendoscopy uses gradient refractive index (GRIN) microlenses that can be inserted into tissue to image cells in deeper areas. This protocol describes in vivo microendoscopy of the mouse hippocampus. The general methodology can be applied to many deep brain regions and other areas of the body.

We present a two-photon microscope that is approximately 2.9 g in mass and 2.0 x 1.9 x 1.1 cm(3) in size and based on a microelectromechanical systems (MEMS) laser-scanning mirror. The microscope has a focusing motor and a micro-optical assembly composed of four gradient refractive index lenses and a dichroic microprism. Fluorescence is captured without the detected emissions reflecting off the MEMS mirror, by use of separate optical fibers for fluorescence collection and delivery of ultrashort excitation pulses. Using this microscope we imaged neocortical microvasculature and tracked the flow of erythrocytes in live mice.

Towards overcoming the size limitations of conventional two-photon fluorescence microscopy, we introduce two-photon imaging based on microelectromechanical systems (MEMS) scanners. Single crystalline silicon scanning mirrors that are 0.75 mm x 0.75 mm in size and driven in two dimensions by microfabricated vertical comb electrostatic actuators can provide optical deflection angles through a range of approximately16 degrees . Using such scanners we demonstrated two-photon microscopy and microendoscopy with fast-axis acquisition rates up to 3.52 kHz.

Fluorescence excitation spectra of fulvic acid (FA) and humic acid (HA) were recorded at different pH as well as at different netural salt concentrations. Spectra of both FA and HA exhibited distinct bands at 465 nm, while spectra of FA showed additional bands at 360 nm. Fluorescence intensities decreased with decreasing pH and increasing ionic strength. This is due to decreasing ionization, increasing particle association, and to coiling of macromolecular structures. The fluorescence characteristics of FA and HA were observed to be related to the free radical content, color, and phenolic structures. Our data show that fluorescence excitation spectra can be utilized for differentiating between soil FA and HA.

Optical approaches for tracking neural dynamics are of widespread interest, but a theoretical framework quantifying the physical limits of these techniques has been lacking. We formulate such a framework by using signal detection and estimation theory to obtain physical bounds on the detection of neural spikes and the estimation of their occurrence times as set by photon counting statistics (shot noise). These bounds are succinctly expressed via a discriminability index that depends on the kinetics of the optical indicator and the relative fluxes of signal and background photons. This approach facilitates quantitative evaluations of different indicators, detector technologies, and data analyses. Our treatment also provides optimal filtering techniques for optical detection of spikes. We compare various types of Ca2+ indicators and show that background photons are a chief impediment to voltage sensing. Thus, voltage indicators that change color in response to membrane depolarization may offer a key advantage over those that change intensity. We also examine fluorescence resonance energy transfer indicators and identify the regimes in which the widely used ratiometric analysis of signals is substantially suboptimal. Overall, by showing how different optical factors interact to affect signal quality, our treatment offers a valuable guide to experimental design and provides measures of confidence to assess optically extracted traces of neural activity.

SummaryMotor units comprise a pre-synaptic motor neuron and multiple post-synaptic muscle fibers. Many movement disorders disrupt motor unit contractile dynamics and the structure of sarcomeres, skeletal muscle’s contractile units. Despite the motor unit’s centrality to neuromuscular physiology, no extant technology can image sarcomere twitch dynamics in live humans. We created a wearable microscope equipped with a microendoscope for minimally invasive observation of sarcomere lengths and contractile dynamics in any major skeletal muscle. By electrically stimulating twitches via the microendoscope and visualizing the sarcomere displacements, we monitored single motor unit contractions in soleus and vastus lateralis muscles of healthy individuals. Control experiments verified that these evoked twitches involved neuromuscular transmission and faithfully reported muscle force generation. In post-stroke patients with spasticity of the biceps brachii, we found involuntary microscopic contractions and sarcomere length abnormalities. The wearable microscope facilitates exploration of many basic and disease-related neuromuscular phenomena never visualized before in live humans.Video Abstract

Recent advances in fluorescence imaging permit studies of Ca(2+) dynamics in large numbers of cells, in anesthetized and awake behaving animals. However, unlike for electrophysiological signals, standardized algorithms for assigning optically recorded signals to individual cells have not yet emerged. Here, we describe an automated sorting procedure that combines independent component analysis and image segmentation for extracting cells' locations and their dynamics with minimal human supervision. In validation studies using simulated data, automated sorting significantly improved estimation of cellular signals compared to conventional analysis based on image regions of interest. We used automated procedures to analyze data recorded by two-photon Ca(2+) imaging in the cerebellar vermis of awake behaving mice. Our analysis yielded simultaneous Ca(2+) activity traces for up to >100 Purkinje cells and Bergmann glia from single recordings. Using this approach, we found microzones of Purkinje cells that were stable across behavioral states and in which synchronous Ca(2+) spiking rose significantly during locomotion.

SummaryMany animals rely on visual motion detection for survival. Motion information is extracted from spatiotemporal intensity patterns on the retina, a paradigmatic neural computation. A phenomenological model, the Hassenstein-Reichardt correlator (HRC), relates visual inputs to neural activity and behavioral responses to motion, but the circuits that implement this computation remain unknown. By using cell-type specific genetic silencing, minimal motion stimuli, and in vivo calcium imaging, we examine two critical HRC inputs. These two pathways respond preferentially to light and dark moving edges. We demonstrate that these pathways perform overlapping but complementary subsets of the computations underlying the HRC. A numerical model implementing differential weighting of these operations displays the observed edge preferences. Intriguingly, these pathways are distinguished by their sensitivities to a stimulus correlation that corresponds to an illusory percept, “reverse phi,” that affects many species. Thus, this computational architecture may be widely used to achieve edge selectivity in motion detection.

Historical milestones in neuroscience have come in diverse forms, ranging from the resolution of specific biological mysteries via creative experimentation to broad technological advances allowing neuroscientists to ask new kinds of questions. The continuous development of tools is driven with a special necessity by the complexity, fragility, and inaccessibility of intact nervous systems, such that inventive technique development and application drawing upon engineering and the applied sciences has long been essential to neuroscience. Here we highlight recent technological directions in neuroscience spurred by progress in optical, electrical, mechanical, chemical, and biological engineering. These research areas are poised for rapid growth and will likely be central to the practice of neuroscience well into the future.

Population codes in the brain have generally been characterized by recording responses from one neuron at a time. This approach will miss codes that rely on concerted patterns of action potentials from many cells. Here we analyze visual signaling in populations of ganglion cells recorded from the isolated salamander retina. These neurons tend to fire synchronously far more frequently than expected by chance. We present an efficient algorithm to identify what groups of cells cooperate in this way. Such groups can include up to seven or more neurons and may account for more than 50% of all the spikes recorded from the retina. These firing patterns represent specific messages about the visual stimulus that differ significantly from what one would derive by single-cell analysis.

Fluctuations in biochemical processes can provide insights into the underlying kinetics beyond what can be gleaned from studies of average rates alone. Historically, analysis of fluctuating transmembrane currents supplied information about ion channel conductance states and lifetimes before single-channel recording techniques emerged. More recently, fluctuation analysis has helped to define mechanochemical pathways and coupling ratios for the motor protein kinesin as well as to probe the contributions of static and dynamic disorder to the kinetics of single enzymes. As growing numbers of assays are developed for enzymatic or folding behaviors of single macromolecules, the range of applications for fluctuation analysis increases. To evaluate specific biochemical models against experimental data, one needs to predict analytically the distribution of times required for completion of each reaction pathway. Unfortunately, using traditional methods, such calculations can be challenging for pathways of even modest complexity. Here, we derive an exact expression for the distribution of completion times for an arbitrary pathway with a finite number of states, using a recursive method to solve algebraically for the appropriate moment-generating function. To facilitate comparisons with experiments on processive motor proteins, we develop a theoretical formalism for the randomness parameter, a dimensionless measure of the variance in motor output. We derive the randomness for motors that take steps of variable sizes or that move on heterogeneous substrates, and then discuss possible applications to enzymes such as RNA polymerase, which transcribes varying DNA sequences, and to myosin V and cytoplasmic dynein, which may advance by variable increments.

SummaryForming distinct representations and memories of multiple contexts and episodes is thought to be a crucial function of the hippocampal-entorhinal cortical network. The hippocampal dentate gyrus (DG) and CA3 are known to contribute to these functions, but the role of the entorhinal cortex (EC) is poorly understood. Here, we show that Ocean cells, excitatory stellate neurons in the medial EC layer II projecting into DG and CA3, rapidly form a distinct representation of a novel context and drive context-specific activation of downstream CA3 cells as well as context-specific fear memory. In contrast, Island cells, excitatory pyramidal neurons in the medial EC layer II projecting into CA1, are indifferent to context-specific encoding or memory. On the other hand, Ocean cells are dispensable for temporal association learning, for which Island cells are crucial. Together, the two excitatory medial EC layer II inputs to the hippocampus have complementary roles in episodic memory.

Neuron activity across the brain How is it that groups of neurons dispersed through the brain interact to generate complex behaviors? Three papers in this issue present brain-scale studies of neuronal activity and dynamics (see the Perspective by Huk and Hart). Allen et al. found that in thirsty mice, there is widespread neural activity related to stimuli that elicit licking and drinking. Individual neurons encoded task-specific responses, but every brain area contained neurons with different types of response. Optogenetic stimulation of thirst-sensing neurons in one area of the brain reinstated drinking and neuronal activity across the brain that previously signaled thirst. Gründemann et al. investigated the activity of mouse basal amygdala neurons in relation to behavior during different tasks. Two ensembles of neurons showed orthogonal activity during exploratory and nonexploratory behaviors, possibly reflecting different levels of anxiety experienced in these areas. Stringer et al. analyzed spontaneous neuronal firing, finding that neurons in the primary visual cortex encoded both visual information and motor activity related to facial movements. The variability of neuronal responses to visual stimuli in the primary visual area is mainly related to arousal and reflects the encoding of latent behavioral states. Science , this issue p. eaav3932 , p. eaav8736 , p. eaav7893 ; see also p. 236

FRET between fluorescent proteins is widely used to monitor biochemical processes in living cells as the length scale of FRET is well matched to protein conformational changes and protein-protein interactions. Although most FRET-based reporters rely on CFPs and YFPs, aspects of CFPs and YFPs are problematic for FRET. CFPs and YFPs can undergo rapid multirate and reversible photobleaching, YFPs can photoconvert into cyan fluorescent species, CFPs can photoactivate at YFP excitation wavelengths and the violet CFP excitation light can be phototoxic. Furthermore, many CFP- and YFP-based FRET reporters produce small changes in FRET, creating detection challenges when the imaged structures are small or when biochemical responses are subtle or transient.In this work, we have developed new fluorescent proteins with properties better suited to a wide range of FRET applications. Starting from Aequorea victoria GFP and the RFP mRuby, we engineered Clover and mRuby2, which conferred greater dynamic range and photostability to four existing FRET reporter designs. Among the improved reporters are a voltage sensor that allows more reliable detection of single action potentials than do previous sensors and an improved RhoA reporter able to detect local and rapid RhoA activation in neuronal growth cones during ephrinA-stimulated retraction.