Figure 3.

In vivo quantification of HbO and HbR changes using tdTomato emission in fiber photometry and correction of GCaMP signals for Hb absorption

(A) Preparation for fiber photometry measurement of GCaMP and tdTomato signals in S1FL.

(B) Confocal images showing GCaMP and tdTomato expression.

(C) GCaMP and tdTomato emission spectra from 488-nm excitation.

(D) Concurrent CBV measurement was achieved by MRI and intravenous injection of long-circulating iron oxide nanoparticles (Feraheme). The inset shows a map of group-level fMRI activation induced by forepaw stimulation (corrected p < 0.05, n = 5 hemispheres).

(E–L) Peri-stimulus heatmaps showing repeated trials and average response time courses of the changes in measured GCaMP, measured tdTomato, derived HbO, derived HbR, derived HbT, corrected GCaMP, corrected tdTomato, and measured MRI-CBV, respectively. The gray-shaded segment indicates the forepaw stimulation period. All color bars use the same unit as the y axis of the time course.

(M) Two-dimensional histogram summarizing the changes in HbT and MRI-CBV in 5 hemispheres and 27 trials. The color bar indicates counts on the log scale.

(N) HbT derived from photometry exhibited higher sensitivity than MRI-CBV (∗∗∗p < 0.001, paired t-test).

(O) Cross-correlation at the trial level between the Z scores of the derived HbT and the Z scores of the MRI-CBV over the 10-s peri-stimulus period.

(P) Scatterplots showing the cross-correlation coefficients (left) and time lag (right) of all trials.

(Q and R) Correction of pseudo-undershoot artifacts in GCaMP time courses of (Q) short (1 s), and (R) long (10 s) stimulations, respectively (∗∗∗p < 0.001, paired t-test).

All error bars represent standard deviation.

See also Figures S6, S9, and S10.