Figure 5.

Brain slice imaging with RhoVR1-PEG₂₅-Halo 15 under widefield, one-photon confocal, and two-photon microscopy. a-i) One-photon, confocal microscopy of 15 in mouse brain slice prepped from animals expressing HaloTag-pDisplay and EGFP (introduced via in utero electroporation). Maximum projection of a) EGFP (green), b) 15 (magenta), and c) merged fluorescence in mouse cortical brain slice stained with 15 (250 nM, 15 min loading). Maximum projections are constructed from 25 slices with optical sections of about 0.8 μm. d-i) Zoomed-in, single optical slice confocal images of cells from panels (a-c). d and g) EGFP-associated fluorescence. e and h) RhoVR-associated fluorescence. f and i) Overlay of EGFP (green) and 15 (magenta) fluorescence. Scale bar is 20 μm. j-k) Two photon microscopy of 15 in cortical brain slice prepared from a mouse expressing pDisplay-HaloTag (in utero electroporation). j) RhoVR fluorescence associated with a slice treated with 250 nM 15 for 15 min at room temperature. Image is a maximum projection of 100 optical slices over 37 μm, with excitation provided at 860 nm. Scale bar is 20 μm. k and 1) Expanded view of boxed regions in panel (j). Scale bar is 10 μm. m-o) Widefield fluorescence microscopy and dual voltage imaging and electrophysiology of cortical neurons in brain slice stained with 15. m) Widefield fluorescence image of 15 fluorescence (250 nM, 15 min loading) in a cortical neuron expressing HaloTag-pDisplay. n) Plot of voltage vs. time for the neuron in panel (n) during current injection to evoke action potentials. Electrophysiology is digitized at 20 kHz. o) Plot of ΔF/F vs. time for the same neuron. Fluorescence data was acquired at 500 Hz, and is not corrected for bleaching. Arrows indicate evoked spike. Small spike are sub-threshold current injections. Scale bar is 20 μm.