Neurophotonics
May 31 – June 7, 2025
Director: Mark Schnitzer
Stanford University, USA
Faculty
Hillel Adesnik, University of California, Berkeley, USA
Na Ji, University of California, Berkeley, USA
Valentina Emiliani, Vision Institute, CNRS, Paris, France
Michael Lin, Stanford University, USA
Mark Schnitzer, Stanford University, USA
Chris Xu, Cornell University, New York, USA
Lin Tian, Max Planck Florida Institute for Neuroscience, Jupiter. USA
The last decade has witnessed a veritable explosion of optical methods for monitoring and manipulating brain activity. New fluorescent indicators, optogenetic actuators of neural dynamics, and microscopes for imaging neural activity patterns at cellular resolution and at large scales have collectively provided neuroscientists with game-changing capabilities for visualizing and experimentally perturbing brain dynamics in awake behaving animals.
This Advanced Course explores these cutting-edge topics in neurophotonics and features tutorial lectures on both foundational principles and the most recent advances in the use of light to study brain dynamics, across scales from single neurons to ensembles of ~100,000 cells or more. The panel of world experts will cover multiple topics, including fluorescent indicators of neural calcium, voltage, neurotransmission and neuromodulatory dynamics; optogenetic excitation and inhibition of neural activity; imaging of electrical oscillations and brain waves; tabletop and head-mounted miniature microscopes that use one-, two- or three-photon fluorescence excitation for imaging neural dynamics in head-fixed or freely behaving animals; adaptive optical, multi-plane, high-speed, multi-beam and volumetric imaging of large-scale neural activity patterns; and holographic and two-photon optogenetic methods for precisely manipulating neural firing.
Overall, the Advanced Course will allow students and scholars presently using or planning to use optical techniques to gain a deeper understanding of the conceptual underpinnings and recent advancements in the rapidly emerging, exciting field of neurophotonics.
Hillel Adesnik
The bioengineering of optimized optogenetic actuators
All optical interrogation of neural circuits enables the precise measurement and recreation of dynamic patterns of neural activity in behaving animals. To understand neural coding and computation, key advances are needed to increase the scale, speed and precision of such optical technologies. To increase the number of optically addressable neurons at a time, I will present our work on the bioengineering of optimized optogenetic actuators, which enable control of much larger populations with the same power budget. To increase the spatial scale,e I will present our development of a mesoscale holographic two-photon microscope for photo-stimulating one cortical area while recording activity across several other areas simultaneously. To address the speed, I will describe a system that combines Bessel beam microscopy with two-photon holographic optogenetics and a novel soma-targeted bifunctional read/write optogenetic tool. This new approach enables the read-out and write-in of population spike trains with single spike precision to recreate natural cortical activity patterns.
Valentina Emiliani
Precise neuronal circuit interrogation using two-photon holographic illumination, optogenetics and voltage imaging
Optogenetic neuronal targeting combined with single-photon wide-field illumination has already demonstrated immense potential in neuroscience, enabling optical control of entire neuronal networks and unravelling their roles in specific behaviours. However, to understand how individual neurons or subsets of neurons control specific behaviours, how functionally identical neurons connect during particular tasks, or how neuronal circuits dynamically modify behaviours in real time, more advanced approaches require more sophisticated approaches enabling the activation of neuronal circuits with single-cell resolution and millisecond temporal accuracy. Over the past few years, this need has driven the development of flexible optical methods for two-photon (2P) optogenetic activation, which enable the interrogation of brain circuits in depth with high spatial and temporal resolution over large volumes. We will review the most commonly used methods for 2P optogenetics, including laser spiral scanning, holographic light multiplexing, and temporal focusing. We will then present the most recent variants of these systems, enabling us to scale up the number of achievable targets through fast sequential light targeting or to reach circuit manipulation in freely moving mice through 2P-holographic endoscopy. Finally, we will demonstrate how the same holographic system used for 2P optogenetics also enables 2P scanless voltage recording from multiple targets in the in vivo mouse brain.
Na Ji
The evolution of spatial and temporal resolution of multiphoton microscopy: Applications to brain imaging
Understanding information processing in the brain requires us to monitor neural activity in vivo at high spatiotemporal resolution. Thanks to a proliferation of genetically encoded fluorescence indicators that monitor diverse neural signaling events, it is now possible to image calcium transients, neurotransmitter and neuromodulator release, and membrane voltage in vivo. Multiphoton fluorescence microscopy is uniquely suited for studying these events in live animals because it can monitor neural signaling at synaptic and cellular resolution in opaque brains at depth. However, the imaging speed of conventional multiphoton microscopy is often not high enough to keep track of fast events such as membrane potentials or to allow measurement of volumetric activity. At large imaging depths, the resolution of multiphoton microscopy is also degraded by brain-induced aberrations, which degrade image quality and can lead to erroneous conclusions on neuronal function. In this talk, I will review our recent work advancing the spatial and temporal resolution of multiphoton microscopy and their applications to brain imaging.
Lin Tian
Fluorescence proteins- and chemical dyes-based genetically encoded indicators for new insights in behaviour and drug discovery
To study neural circuitry, the action of one cell under the context of others, one would precisely measure and perturb specific neuronal populations and molecules in behaving animals specifically engaged in performing the computation or function of interest. The dataset of millions of neurons firing together underlying a given behaviour is required to develop and refine theories (hypotheses) explaining animal behaviour in terms of brain physiology.
One focus of the presentations is how our lab develops novel genetically encoded indicators based on fluorescence proteins and chemical dyes, especially focusing on direct and specific measurement of myriad neurochemical releases (including neurotransmitters, neuromodulators, and neuropeptides) with needed spatial and temporal resolutions. Furthermore, the molecular mechanisms of sensor design, screening, and optimization pipeline based on machine learning, as well as applications to reveal new insights into regulatory roles of neuromodulators in flexible behaviour and drug discovery, will be discussed. In combination with calcium imaging and optogenetics, these sensors are well poised to permit direct functional analysis of how the spatiotemporal coding of neural input signalling mediates the plasticity and function of target circuits.
Michael Lin
From chemistry to cognition: Fluorescent indicators of neuronal activity
Understanding how the biochemical and electrical activities of neurons give rise to the rich functional repertoire of the brain is a fundamental goal of neuroscience. The development of fluorescent indicators for the various facets of neuronal activity, particularly genetically encoded indicators, is driving rapid progress toward this goal. This lecture will explain the features, advantages, and limitations of different fluorophores, the chemical mechanisms by which fluorescent indicators of neuronal activity operate, and the current state of the art in genetically encoded indicators for calcium, voltage, and neurotransmitter release. In addition, we will discuss how the kinetic characteristics of various forms of neuronal activity and the biological organization of circuits influence the choice of optical recording method. In particular, a comparison of parameters governing voltage and calcium imaging provides useful and unexpected insights.
Mark Schnitzer
Fluorescence calcium and voltage imaging techniques for large-scale neural processing
Fluorescence calcium and voltage imaging techniques can reveal the dynamics and coding properties of large ensembles of neurons in behaving animals. I will discuss recent technical developments and biological studies by our group that have used these imaging methods to probe large-scale neural processing of visual scenes, associative memories, and learned movements in behaving mice and flies. One approach to large-scale calcium imaging involves fluorescence mesoscopes with wide fields-of-view. Using this approach, we conducted the first analysis of neural coding across an entire mammalian visual cortex at cellular resolution. To track neural activity concurrently in multiple areas of the brain that may be non-contiguous, we built a robotic multi-arm two-photon microscope, termed the ‘Octopus’, and we are using it for simultaneous imaging of neural activity in up to 4 different brain areas at once. To compare one- and two-photon fluorescence calcium imaging, we built a microscope that acquires one- and two-photon fluorescence signals in near simultaneity from each neuron under view, allowing us to directly evaluate the activity traces for each cell acquired with the two different modalities. Whereas calcium imaging has allowed many thousands of cells to be sampled concurrently, voltage imaging provides superior time resolution and enables reliable detection and sub-millisecond timing estimation accuracy of single action potentials in behaving animals. A recently created suite of 4 mutually compatible voltage indicators based on the Ace opsin enables voltage imaging studies to be performed in behaving animals in up to 4 different neuron types at once. Finally, I will present a scalable computational platform, EXTRACT, based on the framework of robust statistics, for fast and accurate extraction of neural activity signals from large-scale fluorescence movies.
Chris Xu
Pushing the limits of multiphoton microscopy
For over three decades, multiphoton microscopy has created a renaissance in the imaging community. It has changed how we visualize cells by providing high-resolution, non-invasive imaging capability deep within intact tissue. These lectures will first present the history and fundamental physics behind multiphoton imaging. We will then discuss the challenges of deep tissue and high-resolution optical imaging and illustrate the requirements and approaches for imaging the dynamic neuronal activity at the cellular level over a large area and depth in awake and behaving animals. We will further discuss the limits of the imaging depth, volume, and speed in multiphoton imaging of living systems. Finally, we will describe a number of directions for continued development, including new laser sources, new spectral windows, and optimum illumination schemes.