Optical recordings of membrane potential using genetically targeted voltage-sensitive fluorescent proteins

Optical Recording of Action Potentials with Second-Harmonic Generation Microscopy

“…Intrinsic SHG imaging at depths of up to 300 -400 m with submicron resolution has been demonstrated previously in hippocampal brain slices (Dombeck et al, 2003). Because SHG is collected in the transmitted light direction, sample thickness is limited to approximately Ͻ500 m. Staining thick turbid media, such as intact ganglia or mammalian neural tissue, has been accomplished by bath-applying dye (Delaney et al, 1994), but other techniques such as pressure injection of dye (Stosiek et al, 2003), intracellular labeling (Antic et al, 1999), novel GFP constructs (Knopfel et al, 2003), or the addition of dye crystals into tissue (Gan et al, 2000;Moreaux et al, 2001) have been needed previously to stain at these depths. Additionally, varying preparations have led previously to different sensitivities of the same V m dyes (Zochowski et al, 2000), but a greater issue for SHG dyes may be their effects on more delicate mammalian cells.…”

“…Slow transmembrane redistribution of dyes allows for V m imaging with a high signalto-noise ratio (S/N) but cannot provide the 1 msec temporal resolution needed to record fast V m signals (Rink et al, 1980). Elegant green fluorescent protein (GFP) constructs (Knopfel et al, 2003) and fluorescence resonance energy transfer pairs (Gonzalez and Tsien, 1997) have been used to record V m but can be limited either in their response time or in their ability to stain intact tissue (Zochowski et al, 2000), respectively. Methods using intrinsic changes in linear scattering or birefringence have been used to record action potentials (APs) in thin specimens (Cohen et al, 1968;Stepnoski et al, 1991), but recent attention has focused on fluorescent probes.…”

Targeted Optical Probing of Neuronal Circuit Dynamics Using Fluorescent Protein Sensors

“…VSFPs was originally designed to exploit the voltage-dependent conformational changes associated with the voltage sensor domain (VSD) of voltage-gated K+ channel (Kv) proteins and more recently extended to the use of a VSD of ci-VSP [29,30] . VSFPs use either FRET [29] or permuted fluorescent proteins [31] . With the exception of VSFP2.1 and its derivatives, most of the currently available fluorescent protein voltage sensors are poorly targeted to the plasma membrane of neurons, so that a major fraction of the fluorescence is sampled from non-responsive GFP [28,30] .…”

Engineering and characterization of an enhanced fluorescent protein voltage sensor

“…Previous prototypic fluorescent protein voltage sensors were developed by molecular fusion of a GFP-based fluorescent protein to voltage-gated ion channels or components thereof [1][2][3][4]. The first prototype, FlaSh, was obtained by inserting GFP in the Cterminus of the Drosophila Shaker potassium channel [1].…”

“…Another prototype, SPARC, is based on the insertion of GFP into a skeletal muscle sodium channel [4]. Concomitantly, our laboratory explored a different design principle that exploits the voltagedependent conformational changes of an isolated voltage sensor domain [2,3]. The prototype based on this design principle, VSFP1, is composed of the voltage sensor domain from the Kv2.1 potassium channel fused to a pair of cyan and yellow fluorescent proteins (CFP and YFP) [2].…”