Extended Data Fig. 7: Ratiometric two-photon imaging of chemogenetically induced H2O2 dynamics in acute brain slices using the HyPerFLEX-UnaG sensor.
From: A color-tailored fluorogenic sensor for hydrogen peroxide
a, Two-photon excitation fluorescence ratiometric imaging of neurons ex vivo. Images of UnaG (green) and HyPerFLEX-HBR3,5-DOM (red) at depths of 50 µm, 100 µm, 150 µm, 200 µm, and 250 µm below the brain slice surface after fluorogen and D-norvaline addition. b, Depth dependence of two-photon-excited HyPerFLEX signal in brain tissue using HBR-3,5-DOM (orange triangles) and N871b (red circles) fluorogens plotted against tissue depth. The gray line represents typical exponential signal decay due to scattering. c, Intensity dynamics of the sensor’s pseudoratiometric signal. Changes in HBR-3,5-DOM (orange triangles) and UnaG (cyan boxes) fluorescence after fluorogen (fgn arrow) and D-Norvaline (D-Norval arrow) addition in a flow system. d, HyPerFLEX/UnaG fluorescence ratio in control brain slices. Mean (black solid lines) and individual (colored lines) responses of the HyPerFLEX(HBR-3,5-DOM)/UnaG fluorescence ratio at various depths. e, HyPerFLEX/UnaG fluorescence ratio in neurons. Mean (black solid lines) and individual (colored lines) responses from neurons in three brain slices. f, Two-photon excitation fluorescence images. Representative images from the UnaG and HyPerFLEX channels before and after HBR-3,5-DOM fluorogen addition. All images and data represent typical experimental results.
