Primer to Voltage Imaging With ANNINE Dyes and Two-Photon Microscopy

• Published in: Frontiers in cellular neuroscience Vol. 13; p. 321
• Main Authors: Kuhn, Bernd, Roome, Christopher J
• Format: Journal Article
• Language: English
• Published: Switzerland Frontiers Research Foundation 16.07.2019; Frontiers Media S.A
• Subjects: ANNINE; Bleaching; Calcium imaging; Dendrites; Design; Dyes; Electric fields; electrochromism; Electrophysiological recording; Electroporation; Entropy; Hydrocarbons; Hydrogen bonds; Labeling; Lipids; Membrane potential; Microscopy; Nervous system; Neuroscience; Phototoxicity; solvatochromism; Voltage; voltage imaging; voltage-sensitive dye; VSD; Japan; Okinawa; two-photon; voltage imaging; ANNINE; VSD; membrane potential; electrochromism; voltage-sensitive dye; solvatochromism
• ISSN: 1662-5102; 1662-5102
• DOI: 10.3389/fncel.2019.00321

ANNINE-6 and ANNINE-6plus are voltage-sensitive dyes that when combined with two-photon microscopy are ideal for recording of neuronal voltages , in both bulk loaded tissue and the dendrites of single neurons. Here, we describe in detail but for a broad audience the voltage sensing mechanism of fast voltage-sensitive dyes, with a focus on ANNINE dyes, and how voltage imaging can be optimized with one-photon and two-photon excitation. Under optimized imaging conditions the key strengths of ANNINE dyes are their high sensitivity (0.5%/mV), neglectable bleaching and phototoxicity, a linear response to membrane potential, and a temporal resolution which is faster than the optical imaging devices currently used in neurobiology (order of nanoseconds). ANNINE dyes in combination with two-photon microscopy allow depth-resolved voltage imaging in bulk loaded tissue to study average membrane voltage oscillations and sensory responses. Alternatively, if ANNINE-6plus is applied internally, supra and sub threshold voltage changes can be recorded from dendrites of single neurons in awake animals. Interestingly, in our experience ANNINE-6plus labeling is impressively stable , such that voltage imaging from single Purkinje neuron dendrites can be performed for 2 weeks after a single electroporation of the neuron. Finally, to maximize their potential for neuroscience studies, voltage imaging with ANNINE dyes and two-photon microscopy can be combined with electrophysiological recording, calcium imaging, and/or pharmacology, even in awake animals.