These equations can be expressed as a matrix: equation(3) [J]=[S][I],[J]=[S][I],whereby the unmixed image [I] can be calculated using the inverse matrix of S: equation(4) [I]=[S]−1[J].[I]=[S]−1[J]. Assuming the detected signal in both channels represents the total spectral contribution for both fluorophores: equation(5)

s1,1+s2,1=1,s1,1+s2,1=1, equation(6) selleck chemicals s1,2+s2,2=1.s1,2+s2,2=1.[S] was determined experimentally by dual channel acquisition of single excitation two-photon images of cell culture with single fluorophore expression, adjusting laser power and dwell time to achieve photon count levels approximating in vivo signal intensity (Figures S1A–S1B). The mean contribution for each fluorophore

into each channel representing the reference spectra from the acquired images was calculated using Matlab (Mathworks, Natick, MA, USA). These values were subsequently used for spectral linear unmixing of dual channel 16 bit two-photon raw scanner data learn more into an 8 bit RGB image z-stack using Matlab and ImageJ (National Institutes of Health). S measured from in-vivo-labeled samples was similar to the in vitro determined value, and spectral unmixing with either the in vitro or in vivo values yielded essentially the same result (Figures S1A–S1B). Simulations were performed to validate that the 200–250 μm imaging depth used for our data acquisition is well within the signal intensity range, where spectral unmixing can work reliably (see Supplemental already Experimental Procedures and Figures S1C–S1F). For whole-cell dendritic arbor reconstruction and analysis of dendritic morphology, 3D stacks were manually traced in Neurolucida (MicroBrightField, Inc., Williston, VT, USA). The main apical trunk of each cell was excluded from analysis as its orientation was perpendicular to image stacks and thus could not be reconstructed at high resolution. Dendrites are defined as dendritic segments

stretching from one branch point to the next branch point or from one branch point to the branch tip. Dendritic spine and inhibitory synapse tracking and analysis was performed using V3D (Peng et al., 2010). Dendritic spine analysis criteria were as previously described (Holtmaat et al., 2009). Using these scoring criteria, the lack of image volume rotation from imaging session to session may have resulted in some z-projecting dendritic spines being left unscored. This did not influence quantification of spine dynamics due to their low incidence and the fractional scoring. Inhibitory synapses were identified as puncta colocalized to the dendrite of interest with a minimal size of 3×3 or 8–9 clustered pixels (0.56 μm2) with a minimal average signal intensity of at least four times above shot noise background levels.