Simultaneous two-photon imaging of oxygen and blood flow in deep cerebral vessels. Supplementary figure 1

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1 Simultaneous two-photon imaging of oxygen and blood flow in deep cerebral vessels Jérôme Lecoq, Alexandre Parpaleix, Emmanuel Roussakis, Mathieu Ducros, Yannick Goulam Houssen, Sergei A. Vinogradov and Serge Charpak Supplementary figure 1 a b 110,000 XZ XY 100,000 Variance from PMT signal (AU) 90,000 80,000 70,000 c 60, Mean from PMT signal (au) d 00 PO (mm Hg) Ptp-C343 Calibration curve t (ìs) Systemic hemoglobin saturation (%) O % in air 10 (a) Plotting the variance of a single decay against its mean shows that the phosphorescence signal is shot-noise limited as indicated by the linear fit (red line). (b) Point Spread Function (PSF) of the two-photon set-up measured using a fluorescent bead, whose diameter is smaller than the diffraction limited resolution of the optical setup. Scale bar = 1 ìm. (c) PtP-C343 phosphorescence vs PO calibration plot at 36.5 C. (d) Reversible changes of the oxygen content in air, from % to 100% or to 10%, cause increases and decreases in systemic oxygen saturation in the rat hind paw, as measured using pulse oximeter.

2 Supplementary figure Near-surface phosphorescence limits twophoton imaging of oxygen at depth. a Surface 100 µm Vascular point Tissular point b µm 500 µm 600 µm Area ratio (AU) vessel/tissue mw mw 9-3 mw Time (µs) Depth (µm) (a) Left: Phosphorescence signals were measured in a capillary (red arrow) and the nearby tissue (blue arrow) at increasing depths in the olfactory bulb. PtP-C343 and fluorescein were injected I.V. Scale bars = 0 µm. Right: The phosphorescence signal begins to appear in the tissue, devoid of PtP-C343, when the laser is focused at a depth of 300 µm. This signal increases with depth and becomes large at 600 µm. Note that the signal from the vasculature was kept constant at all depths by increasing the excitation laser power. Taking into account the 10% duty cycle, P = 1.1 mw, P = 5.9 mw, 100µm 300µm P = 11.9 mw, P = 0.4 mw. (b) Summary graph of the ratio of vessel/tissue 500µm 600µm phosphorescence signals (three animals). At 300 µm, 90% of photons collected from capillaries are due to the probe in the focal volume. The contribution of surface signal increases significantly at 400 µm. Increase in the laser power (e.g. at 500 ìm) does not improve the ratio.

3 Supplementary figure 3 Simultaneous PO and RBCs flow measurements in all neuronal s of the mouse olfactory bulb. surface (1) glomerular 35 µm /- 0.6 mm Hg (1) PGc () external plexiform 115 µm /- 0.8 mm Hg () (3) Mc (3) mitral cell 05 µm /- 1.4 mm Hg (4) Gc (4) granule cell 70 µm /- 0.8 mm Hg Left: Schematic representation of the olfactory bulb organization. Center: Two-photon imaging of a transgenic mouse expressing eyfp in mitral cells somata and dendrites provides insight on the depth and location of the different neuronal s. YFP cells and processes are shown in green and vessels in red. Scale bar = 0 ìm. Right: At each depth, a capillary is chosen (see white arrow on center column) and both PO and RBCs flow (see inset) are measured. Periglomerular cells (PGc), mitral cells (Mc), granule cells (Gc).

4 Supplementary Methods Animal preparation Animals were held in a standard stereotaxic apparatus, and their body temperature was maintained at 37 C with a feedback-controlled heating blanket (Harvard Apparatus). For all experiments, breathing frequency was monitored through a pneumogram transducer (BIOPAC Systems, Inc.). Heart rate, oxygen saturation and pulse distention were monitored using a pulse oximeter, placed on the rat s hind-paw (STARR Life Sciences). Oxygen saturation was maintained stable above 9-93% with or without supplementary oxygen (<30%). The posterior cisterna was drained. A craniotomy was performed above the two olfactory bulb hemispheres, and the dura was removed. A 100 μm-thick glass coverslip was placed over the bulb, fixed on the cranium, and the space below it was filled with a 3.5% agar solution to limit movements of the brain caused by pulse distention. The oxygen content was controlled by mixing oxygen and nitrogen from tanks, equipped with individual flowmeters (Aalborg). 100 µl of a solution of PtP-C343 (1-5 mm) was injected I.V. within 10 s (the final concentration of PtP-C343 was estimated to be ~ 1/100 of the initial solution). We monitored the cardiovascular state of the animal before and after PtP-C343 injection (oxygen systemic saturation before injection = %, after injection = %; cardiac rate before injection = bpm, after injection = bpm). In some experiments, a bolus of 70 kda fluorescein dextran (Invitrogen) was also injected to provide labeling of microvascular compartments or to measure vascular responses to odor stimulation (see main text). In Vivo Two-Photon PO Microscopy The AOM was controlled by a computer using a home-made high frequency electronic circuit. Scanning patterns for the galvanometric scanners (Cambridge Technology) were synchronized with the AOM gates using home-built electronics and LabVIEW TM software (National Instruments). Laser light was focused onto cerebral blood vessels with a 63 water-immersion objective (Leica). The collected emission was forwarded to a dichroic mirror (cut-off wavelength, 770 nm) and then divided into two channels by another dichroic mirror (cut-off wavelength, 560 nm) (Fig. 1a). The signal in the "green" channel was band-pass-filtered (HQ 50/40m Chroma Technologies Corp), while two shortpass filters (FF01-750/sp-5, SEMROCK) were placed in front of the red-sensitive photomultiplier tube (PMT, R10699, Hamamatsu) - "red" channel. The PMT signals were amplified, integrated by a custom built electronic circuit and sampled at 1.5 MHz by an acquisition card (PCI 605E, MIO16E- 1, National Instruments). The overall system response time was μs.

5 EATs extraction We detected RBCs using the same algorithm developed for RBC flow measurements. We sorted phosphorescence decays according to their distance in time to the nearest RBC. Decays at similar distance were averaged to give oxygen measurements. At the probe concentration of 10 M, ~ 40,000 decays needed to be averaged to reach S.E.M. <5 mm Hg in PO. This corresponds to 0,000 RBCs (two distance measurements per cell, in front of and behind the RBC), or 800 s of continuous recording (Fig. 3d). Given that the average RBC velocity is 0.5 mm s -1, we estimated that the RBC displacement during one ON-OFF cycle is ~ 0.1 m. Since our lateral resolution is < 1 μm (Suppl. Fig. 1b), averaging four successive decays would not affect the measurement. Thus, around 5,000 RBC s or ~ 00 s of flow were recorded in each capillary. To minimize continuous light exposure, the recording was repeated 0 times using 10s-long data collection periods. Data analysis We analyzed all our data using a home-made LabVIEW program. All the analysis tools used in this study were integrated into our main control program, operational during the course of the experiment and providing immediate feedback to the operator. In the entire study, unless otherwise noted, average values are expressed as mean S.E.M.