Figure 2. High-affinity FLII⁸¹E - 1μ glutamate sensor imaging.

Glutamate imaging with simultaneous cortical field recording under (a) control conditions, (b) in the presence of 10 μM GABAzine, and (c) of 10μM GABAzine + 25 μM TBOA. (a, b, & c) (top, left) Bright field image of the cortical slice. Visible in this image are the field recording electrode (left), stimulating electrode (right), local perfusion pipette (top), and a harp string holding the slice in place (bottom). Scale Bar = 250 μM for all images. Approximate positions of cortical layers are indicated in this and all subsequent figures in the lower portion of each bright field image. (top, right) Three individual glutamate FRET sensor images in each case from ≈ 200 ms before stimulation (left), ≈ 80 ms after stimulation (center), and ≈ 1 s after stimulation (right). Arrows indicate exact time of image capture with respect to cortical field recordings. Color scale bar on right indicates fluorescence ratio value in glutamate FRET sensor images. (center) Evoked cortical field recordings performed simultaneously with glutamate images. Dot indicates time of stimulation. (b and c, inset) Early cortical field potentials on an expanded time scale. Note the slower onset and later peak time in the presence of TBOA. (bottom) Normalized ROI analysis of glutamate FRET sensor images. Time scales are identical for field recordings and ROI analysis. (d) Dual channel results for the imaging results shown in parts (a-c). Blue lines represent CFP fluorescence and yellow lines represent Vensu fluorescence.