2.6 Flavoprotein Analysis

For each stimulation intensity, a total of 420 images were collected with the image size of 172 × 130 pixels (8 × 8 binning). The first 5 seconds (20 frames) were used as a baseline. Image analyses was performed using custom software written in MATLAB (Stebbings et al., 2016). The change in fluorescence (ΔF) with stimulation as a function of the baseline fluorescence (F) was used for measurement of activation. The upper layers (layers 1-3) and lower layers (layers 4-6) were analyzed separately (See Figure 1). Input-output curves were constructed for the AC based on the responses to electrical stimulation amplitudes ranging from 25-600 μA. The region of interest measured 1.5 mm from anterior to posterior and was centered over the area of maximum activation. (Llano et al., 2012; Stebbings et al., 2016). The changes in fluorescence over baseline fluorescence maps (Δf/f maps) were overlaid on the raw fluorescence images for identification and localization of anatomic structures.