Fig. 4: In vivo imaging of PTEN in the mouse brain.
From: Genetically encoded biosensor for fluorescence lifetime imaging of PTEN dynamics in the intact brain
a, Top, schematic of IUE followed by in vivo 2pFLIM in the adult mouse brain. Bottom, representative widefield images of fluorescence intensity and FLIM of L2/3 cells expressing G-PTEN. Scale bar, 100 μm. b, Representative high-magnification images of fluorescence intensity and pseudo-colored lifetime of G-PTEN expression in L2/3 soma (top) and dendrite (bottom). Scale bars, 20 μm (soma) and 15 μm (dendrite). c, Quantification of fluorescence lifetime in G-PTEN-expressing neuronal somas (2.20 ± 0.004 ns, n = 142 cells) and dendrites (2.11 ± 0.009 ns, n = 75), in four mice. d, Representative two-photon in vivo images of L2/3 neuronal somas coexpressing G-PTEN, spCas9 and scrambled control gRNA (Scr), IGF1R gRNA or a Tsc2-targeted gRNA. Scale bar, 20 μm. e, Quantification of G-PTEN fluorescence lifetime in L2/3 neuronal somas targeted with scrambled control gRNA (2.17 ± 0.006 ns, n = 56 cells), an IGF1R-targeted gRNA (P = 0.0005, 2.22 ± 0.013 ns, n = 64 cells) or a Tsc2-targeted gRNA (P = 0.0006, 2.22 ± 0.01 ns, n = 40 cells); 4–6 mice per group, NS P = 0.9263. f, Quantification of L2/3 soma area of neurons expressing scrambled gRNA (693.1 ± 22.86 μm2, n = 57 cells), IGF1R gRNA (P = 0.0009, 537.5 ± 12.87 μm2, n = 70 cells) or Tsc2 gRNA (1,652 ± 66.87 μm2, n = 39 cells); 4–6 mice per group. g, Representative two-photon in vivo images of L2/3 neuronal dendrites coexpressing G-PTEN, spCas9 and scrambled control gRNA (Scr), IGF1R gRNA or Tsc2 gRNA. Scale bar, 5 μm. h, Quantification of G-PTEN fluorescence lifetime in L2/3 neurons targeted with scrambled control gRNA (2.07 ± 0.009 ns, n = 45, and 2.04 ± 0.005 ns, n = 94), IGF1R gRNA (2.14 ± 0.016 ns, n = 31, and 2.02 ± 0.015 ns, n = 49) or Tsc2 gRNA (2.07 ± 0.008 ns, n = 65, and 1.91 ± 0.012 ns, n = 120) for their dendritic shafts and spines, respectively. *P = 0.0188. NS P > 0.9999 and P = 0.8882 for Scrambled Shaft compared to Tsc2 gRNA Shaft and for Scrambled Spine compared to IGF1R gRNA Spine, respectively; 4–6 mice per group. i, Quantification of spine density per 10 μm for G-PTEN neurons expressing scrambled control gRNA (5 ± 0.09, n = 35 dendrites), IGF1R-targeted gRNA (P = 0.0197, 2.93 ± 12.87, n = 19 dendrites) or a Tsc2-targeted gRNA (14.8 ± 0.7, n = 35 dendrites); 4–6 mice per group. ‘n’ denotes number of cells unless otherwise noted. Error bars represent the s.e.m. Statistical difference for c was measured using an unpaired two-tailed student t-test. Statistical differences for e, f, h and i were measured using one-way ANOVA followed by post hoc Tukey’s multiple-comparison test. Representative images represent experiments repeated independently at least three times. Significant differences in c, e, f, h and i produced P < 0.0001, unless otherwise stated. ****P < 0.0001.
