In vitro characterization of prototype GluSnFR constructs. (a) Genetic construction of GluSnFR for protein purification (Upper) or mammalian cell surface display (Lower). His-6 is a hexahistidine protein purification tag. Ig-κ is a murine Ig κ-chain V-J2-C signal peptide. (b) Schematic of surface displayed GluSnFR in ligand-free state. (c) In vitro emission spectra changes of soluble GluSnFR_0N0C to 1 mM glutamate in 50 mM Tris buffer (black) or HBSS (red). (d) Titration curves of soluble GluSnFR_0N0C in 50 mM Tris (black triangle) or HBSS (red square). (e) Maximum ratio change of GluSnFR_0N0C (circle) decreases with increasing ionic strength of buffer.

Spatial resolution of glutamate measurement after synaptic release. (a) (Far Left) FRET channel of SuperGluSnFR expressed on the dendritic surface. (Magnification: ×150.) (Scale bar: 10 μm.) Spatially resolved percent ratio change before (Left), during (Middle), and after (Right) a 10-AP, 30-Hz field stimulus in Ringer's (Upper) and with 100 μM TBOA added (Lower). Images are intensity-modulated. Temporal averages of 10 frames are indicated by bars above the traces in 4b, spatially filtered with a 5-pixel wide (466 nm eq.) two-dimensional wiener filter to reduce noise. (b) Averaged ratio change across the total surface of the expressing neuron in a in Ringer's (black) or with TBOA (red). (c) Spatially resolved ratio change to average of 30 single AP stimuli. (Far Left) FRET as in a (Upper Left to Lower Right) sequential frames of response (33 ms per frame). Stimulation of one AP occurs with slight jitter between frames two and three. (d) Time course of average dendritic response.

Time course of glutamate release and uptake. (a) Average responses to AP (black squares), 10 AP–15 Hz (red circles) and 10 AP–30 Hz (blue triangles) field stimulation in Ringer's solution (solid line) or with 100 μM TBOA added (open shapes, dotted line) to block active glutamate uptake. (b) The decay phase of a scaled to the maximum height to highlight the relative glutamate clearance rate. Colors are as in a. (c) Comparison of surface glutamate in model to observed levels. Average SuperGluSnFR responses to field stimulation in Ringer's solution or in 100 μM TBOA (black). Model of instantaneous glutamate levels in plane of neurons (gray). Model glutamate filtered by the camera integration (red). (d) Model of glutamate double-bound NR2A (black), NR2B (blue), and NR2D (red) NMDA receptors in response to 1 AP, 10 AP–15 Hz, and 10 AP–30 Hz stimulation in Ringer's.

Optimization of GluSnFR response on mammalian cell surface. (a) Amino acid sequence of GltI N-terminal (Upper) and C-terminal (Lower) truncations between ECFP and Citrine. Truncation sites are indicated with thin vertical lines. The truncations for the original GluSnFR_0N0C and best responding construct GluSnFR_8N5C are noted in bold lines. (b) FRET channel showing clean extracellular membrane expression on transfected HEK293 cells. (c) Map of the truncation combination to response magnitude. Color indicates the percentage of maximal ratio change relative to GluSnFR_8N5C. Black indicates improper construct folding. (d) (Left) HEK293 cells transfected with the nonoptimized GluSnFR_0N0C. (Right) The best responder GluSnFR_8N5C. Color bar values encompass the same range of relative CFP/YFP change for both constructs. (e) Ratio change between glutamate free and 100 μM glutamate solutions of HBSS for GluSnFR_0N0C (n = 9 fields) and GluSnFR_8N5C (n = 18). (f) In vitro emission spectra changes of soluble GluSnFR_8N5C to 1 mM glutamate in 50 mM Tris buffer (black) or HBSS (red). (g) Glutamate titration curves of GluSnFR_8N5C HEK/HeLa cells in Ringer's (squares and black dashed line; n = 20) and neurons in Ringer's plus 100 μM TBOA (diamonds and red line; n = 5). (Scale bars: 10 μm.)

Temporal resolution of SuperGluSnFR response. (a) Normalized CFP/YFP emission ratio of a SuperGluSnFR expressing dendritic arbor during single action potential field stimulus. Individual traces are in gray; average traces are in black (n = 27). (b) Corresponding glutamate concentration measurements after calibration.