Neurophotonics beyond the Surface: Unmasking the Brain's Complexity Exploiting Optical Scattering.

The intricate nature of the brain necessitates the application of advanced probing techniques to comprehensively study and understand its working mechanisms. Neurophotonics offers minimally invasive methods to probe the brain using optics at cellular and even molecular levels. However, multiple challenges persist, especially concerning imaging depth, field of view, speed, and biocompatibility. A major hindrance to solving these challenges in optics is the scattering nature of the brain. This perspective highlights the potential of complex media optics, a specialized area of study focused on light propagation in materials with intricate heterogeneous optical properties, in advancing and improving neuronal readouts for structural imaging and optical recordings of neuronal activity. Key strategies include wavefront shaping techniques and computational imaging and sensing techniques that exploit scattering properties for enhanced performance. We discuss the potential merger of the two fields as well as potential challenges and perspectives toward longer term in vivo applications.

Neurophotonics beyond the surface: unmasking the brain's complexity exploiting optical scattering.

The intricate nature of the brain necessitates the application of advanced probing techniques to comprehensively study and understand its working mechanisms. Neurophotonics offers minimally invasive methods to probe the brain using optics at cellular and even molecular levels. However, multiple challenges persist, especially concerning imaging depth, field of view, speed, and biocompatibility. A major hindrance to solving these challenges in optics is the scattering nature of the brain. This perspective highlights the potential of complex media optics, a specialized area of study focused on light propagation in materials with intricate heterogeneous optical properties, in advancing and improving neuronal readouts for structural imaging and optical recordings of neuronal activity. Key strategies include wavefront shaping techniques and computational imaging and sensing techniques that exploit scattering properties for enhanced performance. We discuss the potential merger of the two fields as well as potential challenges and perspectives toward longer term in vivo applications.

Acousto-optic holography for pseudo-two-dimensional dynamic light patterning.

Optical systems use acousto-optic deflectors (AODs) mostly for fast angular scanning and spectral filtering of laser beams. However, AODs may transform laser light in much broader ways. When time-locked to the pulsing of low repetition rate laser amplifiers, AODs permit the holographic reconstruction of 1D and pseudo-two-dimensional (ps2D) intensity objects of rectangular shape by controlling the amplitude and phase of the light field at high (20-200 kHz) rates for microscopic light patterning. Using iterative Fourier transformations (IFTs), we searched for AOD-compatible holograms to reconstruct the given ps2D target patterns through either phase-only or complex light field modulation. We previously showed that phase-only holograms can adequately render grid-like patterns of diffraction-limited points with non-overlapping diffraction orders, while side lobes to the target pattern can be cured with an apodization mask. Dense target patterns, in contrast, are typically encumbered by apodization-resistant speckle noise. Here, we show the denoised rendering of dense ps2D objects by complex acousto-optic holograms deriving from simultaneous optimization of the amplitude and phase of the light field. Target patterns lacking ps2D symmetry, although not translatable into single holograms, were accessed by serial holography based on a segregation into ps2D-compatible components. The holograms retrieved under different regularizations were experimentally validated in an AOD random-access microscope. IFT regularizations characterized in this work extend the versatility of acousto-optic holography for fast dynamic light patterning.

Fast wavefront shaping for two-photon brain imaging with multipatch correction.

Nonlinear fluorescence microscopy promotes in-vivo optical imaging of cellular structure at diffraction-limited resolution deep inside scattering biological tissues. Active compensation of tissue-induced aberrations and light scattering through adaptive wavefront correction further extends the accessible depth by restoring high resolution at large depth. However, those corrections are only valid over a very limited field of view within the angular memory effect. To overcome this limitation, we introduce an acousto-optic light modulation technique for fluorescence imaging with simultaneous wavefront correction at pixel scan speed. Biaxial wavefront corrections are first learned by adaptive optimization at multiple locations in the image field. During image acquisition, the learned corrections are then switched on the fly according to the position of the excitation focus during the raster scan. The proposed microscope is applied to in vivo transcranial neuron imaging and demonstrates multi-patch correction of thinned skull-induced aberrations and scattering at 40-kHz data acquisition speed.

Fast optical recording of neuronal activity by three-dimensional custom-access serial holography.

Optical recording of neuronal activity in three-dimensional (3D) brain circuits at cellular and millisecond resolution in vivo is essential for probing information flow in the brain. While random-access multiphoton microscopy permits fast optical access to neuronal targets in three dimensions, the method is challenged by motion artifacts when recording from behaving animals. Therefore, we developed three-dimensional custom-access serial holography (3D-CASH). Built on a fast acousto-optic light modulator, 3D-CASH performs serial sampling at 40 kHz from neurons at freely selectable 3D locations. Motion artifacts are eliminated by targeting each neuron with a size-optimized pattern of excitation light covering the cell body and its anticipated displacement field. Spike rates inferred from GCaMP6f recordings in visual cortex of awake mice tracked the phase of a moving bar stimulus with higher spike correlation between intra compared to interlaminar neuron pairs. 3D-CASH offers access to the millisecond correlation structure of in vivo neuronal activity in 3D microcircuits.

Fast spatial beam shaping by acousto-optic diffraction for 3D non-linear microscopy.

Acousto-optic deflection (AOD) devices offer unprecedented fast control of the entire spatial structure of light beams, most notably their phase. AOD light modulation of ultra-short laser pulses, however, is not straightforward to implement because of intrinsic chromatic dispersion and non-stationarity of acousto-optic diffraction. While schemes exist to compensate chromatic dispersion, non-stationarity remains an obstacle. In this work we demonstrate an efficient AOD light modulator for stable phase modulation using time-locked generation of frequency-modulated acoustic waves at the full repetition rate of a high power laser pulse amplifier of 80 kHz. We establish the non-local relationship between the optical phase and the generating acoustic frequency function and verify the system for temporal stability, phase accuracy and generation of non-linear two-dimensional phase functions.

Route to genetically targeted optical electrophysiology: development and applications of voltage-sensitive fluorescent proteins.

The invention of membrane voltage protein indicators widens the reach of optical voltage imaging in cell physiology, most notably neurophysiology, by enabling membrane voltage recordings from genetically defined cell types in chronic and life-long preparations. While the last years have seen a dramatic improvement in the technical performance of these indicators, concomitant innovations in optogenetics, optical axon tracing, and high-speed digital microscopy are beginning to fulfill the age-old vision of an all-optical analysis of neuronal circuits, reaching beyond the limits of traditional electrode-based recordings. We will present our personal account of the development of protein voltage indicators from the pioneering days to the present state, including their applications in neurophysiology that has inspired our own work for more than a decade.

Two-photon voltage imaging using a genetically encoded voltage indicator.

Voltage-sensitive fluorescent proteins (VSFPs) are a family of genetically-encoded voltage indicators (GEVIs) reporting membrane voltage fluctuation from genetically-targeted cells in cell cultures to whole brains in awake mice as demonstrated earlier using 1-photon (1P) fluorescence excitation imaging. However, in-vivo 1P imaging captures optical signals only from superficial layers and does not optically resolve single neurons. Two-photon excitation (2P) imaging, on the other hand, has not yet been convincingly applied to GEVI experiments. Here we show that 2P imaging of VSFP Butterfly 1.2 expresssing pyramidal neurons in layer 2/3 reports optical membrane voltage in brain slices consistent with 1P imaging but with a 2-3 larger ΔR/R value. 2P imaging of mouse cortex in-vivo achieved cellular resolution throughout layer 2/3. In somatosensory cortex we recorded sensory responses to single whisker deflections in anesthetized mice at full frame video rate. Our results demonstrate the feasibility of GEVI-based functional 2P imaging in mouse cortex.

Genetically engineered fluorescent voltage reporters.

Fluorescent membrane voltage indicators that enable optical imaging of neuronal circuit operations in the living mammalian brain are powerful tools for biology and particularly neuroscience. Classical voltage-sensitive dyes, typically low molecular-weight organic compounds, have been in widespread use for decades but are limited by issues related to optical noise, the lack of generally applicable procedures that enable staining of specific cell populations, and difficulties in performing imaging experiments over days and weeks. Genetically encoded voltage indicators (GEVIs) represent a newer alternative that overcomes several of the limitations inherent to classical voltage-sensitive dyes. We critically review the fundamental concepts of this approach, the variety of available probes and their state of development.

Imaging neural circuit dynamics with a voltage-sensitive fluorescent protein.

Population signals from neuronal ensembles in cortex during behavior are commonly measured with EEG, local field potential (LFP), and voltage-sensitive dyes. A genetically encoded voltage indicator would be useful for detection of such signals in specific cell types. Here we describe how this goal can be achieved with Butterfly, a voltage-sensitive fluorescent protein (VSFP) with a subthreshold detection range and enhancements designed for voltage imaging from single neurons to brain in vivo. VSFP-Butterfly showed reliable membrane targeting, maximum response gain around standard neuronal resting membrane potential, fast kinetics for single-cell synaptic responses, and a high signal-to-noise ratio. Butterfly reports excitatory postsynaptic potentials (EPSPs) in cortical neurons, whisker-evoked responses in barrel cortex, 25-Hz gamma oscillations in hippocampal slices, and 2- to 12-Hz slow waves during brain state modulation in vivo. Our findings demonstrate that cell class-specific voltage imaging is practical with VSFP-Butterfly, and expand the genetic toolbox for the detection of neuronal population dynamics.

A comprehensive concept of optogenetics.

Fundamental questions that neuroscientists have previously approached with classical biochemical and electrophysiological techniques can now be addressed using optogenetics. The term optogenetics reflects the key program of this emerging field, namely, combining optical and genetic techniques. With the already impressively successful application of light-driven actuator proteins such as microbial opsins to interact with intact neural circuits, optogenetics rose to a key technology over the past few years. While spearheaded by tools to control membrane voltage, the more general concept of optogenetics includes the use of a variety of genetically encoded probes for physiological parameters ranging from membrane voltage and calcium concentration to metabolism. Here, we provide a comprehensive overview of the state of the art in this rapidly growing discipline and attempt to sketch some of its future prospects and challenges.

Genetically encoded probes for optical imaging of brain electrical activity.

The combination of optical imaging methods with targeted expression of protein-based fluorescent probes constitutes a powerful approach for functional analysis of selected cell populations within intact neuronal circuitries. Herein, we lay out the conceptual motivation for optogenetic recording of brain electrical activity using genetically encoded voltage-sensitive fluorescent proteins (VSFPs), describe how the current generation of VSFPs has evolved, and demonstrate how VSFPs report membrane voltage signals in isolated cells, brain slices, and living animals. We conclude with a critical appraisal of VSFPs for voltage recording and highlight promising applications of this emerging methodology for bridging cellular and intact systems biology.

Optogenetic monitoring of membrane potentials.

Over the last decade, researchers in our laboratory have engineered and developed several series of genetically encoded voltage-sensitive fluorescent proteins (VSFPs) by molecular fusion of a voltage-sensing domain operand with different fluorescent reporter proteins. These genetically encoded VSFPs have been shown to provide a reliable optical report of membrane potential from targeted neurons and muscle cells in culture or in living animals. However, these various reporters also exhibit discrepancies in both their voltage-sensing and targeting properties that are essentially related to the intrinsic characteristics of the fluorescent reporter proteins. It is therefore important carefully to select the sensor that is most appropriate for the particular question being investigated experimentally. Here we examine the current state of this subfield of optogenetics, address current limitations and challenges, and discuss what is likely to be feasible in the near future.

Biophysical characterization of the fluorescent protein voltage probe VSFP2.3 based on the voltage-sensing domain of Ci-VSP.

A voltage sensitive phosphatase was discovered in the ascidian Ciona intestinalis. The phosphatase, Ci-VSP, contains a voltage-sensing domain homologous to those known from voltage-gated ion channels, but unlike ion channels, the voltage-sensing domain of Ci-VSP can reside in the cell membrane as a monomer. We fused the voltage-sensing domain of Ci-VSP to a pair of fluorescent reporter proteins to generate a genetically encodable voltage-sensing fluorescent probe, VSFP2.3. VSFP2.3 is a fluorescent voltage probe that reports changes in membrane potential as a FRET (fluorescence resonance energy transfer) signal. Here we report sensing current measurements from VSFP2.3, and show that VSFP2.3 carries 1.2 e sensing charges, which are displaced within 1.5 ms. The sensing currents become faster at higher temperatures, and the voltage dependence of the decay time constants is temperature dependent. Neutralization of an arginine in S4, previously suggested to be a sensing charge, and measuring associated sensing currents indicate that this charge is likely to reside at the membrane-aqueous interface rather than within the membrane electric field. The data presented give us insights into the voltage-sensing mechanism of Ci-VSP, which will allow us to further improve the sensitivity and kinetics of the family of VSFP proteins.

Imaging brain electric signals with genetically targeted voltage-sensitive fluorescent proteins.

Cortical information processing relies on synaptic interactions between diverse classes of neurons with distinct electrophysiological and connection properties. Uncovering the operational principles of these elaborate circuits requires the probing of electrical activity from selected populations of defined neurons. Here we show that genetically encoded voltage-sensitive fluorescent proteins (VSFPs) provide an optical voltage report from targeted neurons in culture, acute brain slices and living mice. By expressing VSFPs in pyramidal cells of mouse somatosensory cortex, we also demonstrate that these probes can report cortical electrical responses to single sensory stimuli in vivo. These protein-based voltage probes will facilitate the analysis of cortical circuits in genetically defined cell populations and are hence a valuable addition to the optogenetic toolbox.

Contribution of plasma membrane Ca ATPase to cerebellar synapse function.

The cerebellum expresses one of the highest levels of the plasma membrane Ca(2+) ATPase, isoform 2 in the mammalian brain. This highly efficient plasma membrane calcium transporter protein is enriched within the main output neurons of the cerebellar cortex; i.e. the Purkinje neurons (PNs). Here we review recent evidence, including electrophysiological and calcium imaging approaches using the plasma membrane calcium ATPase 2 (PMCA2) knockout mouse, to show that PMCA2 is critical for the physiological control of calcium at cerebellar synapses and cerebellar dependent behaviour. These studies have also revealed that deletion of PMCA2 throughout cerebellar development in the PMCA2 knockout mouse leads to permanent signalling and morphological alterations in the PN dendrites. Whilst these findings highlight the importance of PMCA2 during cerebellar synapse function and development, they also reveal some limitations in the use of the PMCA2 knockout mouse and the need for additional experimental approaches including cell-specific and reversible manipulation of PMCAs.

Optical imaging as a link between cellular neurophysiology and circuit modeling.

The relatively simple and highly modular circuitry of the cerebellum raised expectations decades ago that a realistic computational model of cerebellar circuit operations would be feasible, and prove insightful for unraveling cerebellar information processing. To this end, the biophysical properties of most cerebellar cell types and their synaptic connections have been well characterized and integrated into realistic single cell models. Furthermore, large scale models of cerebellar circuits that extrapolate from single cell properties to circuit dynamics have been constructed. While the development of single cell models have been constrained by microelectrode recordings, guidance and validation as these models are scaled up to study network interactions requires an experimental methodology capable of monitoring cerebellar dynamics at the population level. Here we review the potential of optical imaging techniques to serve this purpose.

Second and third generation voltage-sensitive fluorescent proteins for monitoring membrane potential.

Over the last decade, optical neuroimaging methods have been enriched by engineered biosensors derived from fluorescent protein (FP) reporters fused to protein detectors that convert physiological signals into changes of intrinsic FP fluorescence. These FP-based indicators are genetically encoded, and hence targetable to specific cell populations within networks of heterologous cell types. Among this class of biosensors, the development of optical probes for membrane potential is both highly desirable and challenging. A suitable FP voltage sensor would indeed be a valuable tool for monitoring the activity of thousands of individual neurons simultaneously in a non-invasive manner. Previous prototypic genetically-encoded FP voltage indicators achieved a proof of principle but also highlighted several difficulties such as poor cell surface targeting and slow kinetics. Recently, we developed a new series of FRET-based Voltage-Sensitive Fluorescent Proteins (VSFPs), referred to as VSFP2s, with efficient targeting to the plasma membrane and high responsiveness to membrane potential signaling in excitable cells. In addition to these FRET-based voltage sensors, we also generated a third series of probes consisting of single FPs with response kinetics suitable for the optical imaging of fast neuronal signals. These newly available genetically-encoded reporters for membrane potential will be instrumental for future experimental approaches directed toward the understanding of neuronal network dynamics and information processing in the brain. Here, we review the development and current status of these novel fluorescent probes.