Spectral fiber photometry derives hemoglobin concentration changes for accurate measurement of fluorescent sensor activity

Figures

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  Figure 1. Spectrally resolved fiber photometry system and Hb absorption effects (A) Schematic of a spectrally resolved fiber photometry system used for recording fluorescence signals. (B) Hb in red blood cells absorbs light in two phases: excitation light from the optical fiber (left) and emission light to be recorded by the optical fiber (right). The color density of arrowheads indicates light intensity. An increase in Hb is expected to decrease the number of photons in both phases. (C) Simulated average excitation and emission photon traveling path lengths across 488- 700-nm emission wavelengths from our database of paired excitation/emission light paths. (D) Effect of HbO and HbR concentration on ΔF/F. Shown are simulated effects of HbO and HbR concentration changes on measurement of fluorescence signals. ΔCHb0 and ΔCHbR ranging from −90 to +90 μM were used to simulate fluorescence signal changes (ΔF/F) between 500 and 700 nm using the molar extinction coefficients of HbO and HbR (Figure S1) and simulated light paths (Figure 1C). Graphs shaded in blue indicate an overall decrease in total Hb (HbT), and graphs shaded in red indicate an overall increase in HbT. See also Figures S1, S2,…

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  Figure 2. In vivo quantification of HbO and HbR changes using EYFP emission in fiber photometry and correction of EYFP signals for Hb absorption (A) Preparation for fiber photometry measurement of EYFP signals in S1FL. (B) Confocal image showing EYFP expression. (C) Simultaneous dual spectral photometry recording of EYFP signals and hemodynamic responses (CBV) was achieved by intravenously injecting a long-circulating red fluorescent dye, Rhodamine B. (D–J) Peri-stimulus heatmaps showing repeated trials and average response time courses of the changes in measured rhoCBV, measured EYFP, derived HbO, derived HbR, corrected rhoCBV, corrected EYFP, and derived HbT, respectively. The gray-shaded segment indicates the forepaw stimulation period. Hb absorption induces pseudo-negative changes in measured EYFP signals during stimulation. (K and L) Correction of the contaminated EYFP and rhoCBV signals, respectively, in 5 hemispheres and 30 trials (∗∗∗p < 0.001, paired t test). Measured and corrected EYFP signals are compared. Measured rhoCBV was normalized to 0 to show the difference before and after correction. All color bars use the same unit as the y axis of the time course. Error bars represent…

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  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…

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  Figure 4. Hb absorption effects on sustained GCaMP signal measurement using fiber photometry (A) Preparation for fiber photometry measurement of GCaMP and tdTomato signals in the PrL region. (B) Measured and corrected tdTomato time courses before and after ethanol administration. The same volume of saline was administered in a separate session to generate vehicle control data within the subject. (C) Measured and corrected GCaMP time courses before and after ethanol administration. The same volume of saline was administered in a separate session to generate vehicle control data within the subject. Data were collected concurrently with those shown in (B). (D) Derived HbO, HbR, and HbT time courses before and after ethanol administration. Higher HbT Z scores were attributed to the lower baseline variation after addition of HbO and HbR. (E) Measured and corrected tdTomato signals were sampled and displayed at 5 min. A significant effect was found between the three groups, and a post hoc test showed a significant difference between the measured and corrected signals. ∗∗∗p < 0.001, two-way ANOVA (n = 3 subjects). Error bars represent standard deviation. (F) Measured and corrected GCaMP signals…

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  Figure 5. Correction of Hb absorption artifacts in other fiber photometry settings (A) Preparation for fiber photometry measurement of GCaMP and tdTomato signals in the left M1, with the right M1 receiving local micro-electrical stimulation. (B) Interleaved acquisition paradigm with 405- and 488-nm excitation lasers. Ca2+-independent GCaMP signals (GCaMP405) were excited by a 405-nm laser, whereas Ca2+-dependent GCaMP signals (GCaMP488) and tdTomato signals were excited by a 488-nm laser. This interleaved paradigm was used because GCaMP405 and GCaMP488 share nearly identical emission spectra and cannot be spectrally unmixed. Data were acquired at 50% duty cycle every 50 ms using a box paradigm to avoid spectrometer frame loss during trigger mode. The final sampling rate is 10 Hz for all signals. (C) Measured GCaMP488 and GCaMP405 signals without any correction. (D) Corrected GCaMP488 using HbO and HbR dynamics derived from the full GCaMP405 spectrum. (E) Corrected GCaMP488 using HbO and HbR dynamics from 3 datapoints in the GCaMP405 spectrum (at 502, 523, and 544 nm). (F) Corrected GCaMP488 by regressing out the GCaMP405 signal dynamics. (G) Comparisons of the post-stimulus undershoot…

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  Figure 6. Regional differences in brain HRF (A) Preparation for fiber photometry measurement of GCaMP and tdTomato signals in the left M1 and right CPu. Both have a direct anatomical connection with the right M1 receiving local micro-electrical stimulation. (B) Confocal images showing expression of GCaMP and tdTomato in the M1 and CPu. (C and D) Peri-stimulus heatmaps and response time courses of the changes in corrected GCaMP, derived HbO, derived HbR, and derived HbT and modeled HRFs in the M1 and CPu, respectively. Pie charts in the insets indicate the ratio between positive (red) and negative (blue) HRF polarities. All pre-stimulus heatmap color bars use the same unit as the time course y axis. Error bars represent standard deviation (4 subjects, 12 trials). See also Figure S8.