Two-photon microscope for multisite microphotolysis of caged neurotransmitters in acute brain slices

Abstract

Abstract. We developed a two-photon microscope optimized for physiologically manipulating single neurons through their postsynaptic receptors. The optical layout fulfills the stringent design criteria required for high-speed, high-resolution imaging in scattering brain tissue with minimal photodamage. We detail the practical compensation of spectral and temporal dispersion inherent in fast laser beam scanning with acousto-optic deflectors, as well as a set of biological protocols for visualizing nearly diffraction-limited structures and delivering physiological synaptic stimuli. The microscope clearly resolves dendritic spines and evokes electrophysiological transients in single neurons that are similar to endogenous responses. This system enables the study of multisynaptic integration and will assist our understanding of single neuron function and dendritic computation.

From One‐Photon to Two‐Photon Probes: “Caged” Compounds, Actuators, and Photoswitches

“…This can be done either with fast scanning in two spatial dimensions or with holographic excitation in three dimensions. Patterned uncaging of caged glutamate in combination with electrophysiology has been performed in the preparation of brain slices either by using one‐72 or two‐photon excitation,73, 74 and by using a spatial light modulator (SLM) based scanless TP microscope 73. The scanning methods with two‐photon excitation have successfully shown the way in which spatiotemporal integration can generate nonlinear responses to different input patterns 40a,b.…”

Optical developments for optogenetics

“…Resonant scanners have been used mainly for 2P calcium imaging (Fan et al, 1999;Nguyen et al, 2001;Rochefort et al, 2009), whereas AOD scanners have been widely used in 2D for uncaging applications (Shoham et al, 2005;Losavio et al, 2009) and 2P calcium imaging (Salomé et al, 2006;Otsu et al, 2008), also including three dimensions (3D) (Reddy and Saggau, 2005;Reddy et al, 2008;Katona et al, 2012). In addition, investigators recently used AODs to photostimulate optogenetic actuators (Wang et al, 2011), specifically manipulating the activity of neurons expressing the ameliorated chimera variant of channelrhodopsin ChIEF (Lin et al, 2009), achieving high temporal resolution with single-photon excitation (1PE).…”

Von Ein‐ zu Zwei‐Photonen‐Sonden: photoaktivierbare Reagentien, Aktuatoren und Photoschalter

“…Dies kann entweder durch schnelles Abtasten in zwei Raumrichtungen oder durch holographische Anregung in drei Dimensionen realisiert werden. Zum Beispiel wurde die Photofreisetzung von Glutamat mit elektrophysiologischen Verfahren kombiniert, um mittels Ein- [72] oder Zwei-Photonen-Anregung [73,74] und unter Einsatz eines räumlichen Lichtmodulators (SLM) Studien an Gehirnschnitten durchzuführen. [73] Solche Rastermethoden in Kombination mit Zwei-Photonen-Anregung erçffneten auch Mçglichkeiten für die Untersuchung von nichtlinearen Antworten auf synaptische Signalmuster.…”