Simultaneous two-photon imaging of oxygen and blood flow in deep cerebral vessels - PubMed (original) (raw)
Simultaneous two-photon imaging of oxygen and blood flow in deep cerebral vessels
Jérôme Lecoq et al. Nat Med. 2011.
Abstract
Uncovering principles that regulate energy metabolism in the brain requires mapping of partial pressure of oxygen (PO(2)) and blood flow with high spatial and temporal resolution. Using two-photon phosphorescence lifetime microscopy (2PLM) and the oxygen probe PtP-C343, we show that PO(2) can be accurately measured in the brain at depths up to 300 μm with micron-scale resolution. In addition, 2PLM allowed simultaneous measurements of blood flow and of PO(2) in capillaries with less than one-second temporal resolution. Using this approach, we detected erythrocyte-associated transients (EATs) in oxygen in the rat olfactory bulb and showed the existence of diffusion-based arterio-venous shunts. Sensory stimulation evoked functional hyperemia, accompanied by an increase in PO(2) in capillaries and by a biphasic PO(2) response in the neuropil, consisting of an 'initial dip' and a rebound. 2PLM of PO(2) opens new avenues for studies of brain metabolism and blood flow regulation.
Conflict of interest statement
COMPETING FINANCIAL INTERESTS
The authors declare no competing financial interests.
Figures
Figure 1
Measurements of PO2 in deep cerebral vessels using probe PtP-C343. (a) Experimental setup. An AOM is placed in the excitation path of a standard two-photon microscope, enabling fast repetitive on-off switching of the laser excitation. (b) The probe is excited by a brief gate (2.5 μs) of femtosecond pulses from a Ti:sapphire laser (_λ_ex = 850 nm, <250 fs, 76 MHz), followed by a phosphorescence detection period (~250 μs). The fluorescence emitted by PtP-C343 is detected by a photomultiplier tube (PMT) in the green channel (PMT1); the phosphorescence is detected by PMT2 in the red channel. (c) Top, intravenous (i.v.) injection of PtP-C343 and fast scanning with detection of fluorescence reveals capillary in the rat olfactory bulb. Phosphorescence is measured at selected spots (red in the capillary lumen). Scale bar, 10 μm. Bottom, successive phosphorescence acquisition cycles displayed as an image. Each horizontal line is one complete cycle, whereas the vertical axis shows successive cycles. (d) Phosphorescence decay obtained by averaging of 40,000 excitation gates, fitted to a single exponential (red). (e) Respiratory arrest (RA) induced by air injection into the femoral vein. (f) Effect of changes in oxygen content in the inhaled air from 21% to 100% or 10% on PO2. Error bars indicate s.e.m.
Figure 2
Temporal and spatial resolution of two-photon phosphorescence measurement. (a) Log-log plots of the phosphorescence integrated intensity (area under the decay curve after subtraction of the baseline) versus average excitation power. Left, AOM gate 2.5 μs. Maximal power shown: 275 mW after the objective, or 2.7 mW, taking into account 1% duty cycle; focal spot depth 80 μm. Red line (slope = 2) corresponds to pure quadratic dependence. The black arrow indicates the region of quadratic dependence (up to 50–60 mW). Right, AOM gate 25 μs (average laser power ~0.5–0.6 mW, 10% duty cycle). The phosphorescence emission is outside the quadratic regime throughout the entire power range. AU, arbitrary units. (b) Precision of the lifetime (τ) measurement as a function of the number of averages, their duration and PtP-C343 concentration (injected i.v.). Left, AOM gate: 2.5 μs (PtP-C343 concentration: ~10 μM, average laser power ~0.5–0.6 mW). Lifetime measurements were performed in eight capillaries at ~100 μm depth. 12,000 averages (~3 s) are sufficient to reach the desired precision. Right, AOM gate 25 μs (PtP-C343 concentration ~50 μM, average laser power ~7 mW). 3,000 gates (~0.80 s) are sufficient to achieve the same precision as in left. (c) Spatial confinement of phosphorescence measurements under different excitation regimes (left, AOM gate 2.5 μs; right, AOM gate 25 μs). Point measurements were performed perpendicularly to the longitudinal axis of two capillaries. Phosphorescence decays were observed in the capillary lumen (red dots) but not in the neuropil (blue dots). Measurements at capillary boundaries produced weak signal (purple dots). Scale bars, 5 μm.
Figure 3
Simultaneous measurements of RBC flow and PO2 using fluorescence and phosphorescence of PtP-C343. (a) RBC detection. The fluorescence of PtP-C343 in the plasma during the excitation gate is shadowed by passing RBCs (white arrows on right pictures corresponding to individual schematics on the left). (b,c) Automatic detection of RBC transients (b) allows extraction of local RBC flow rates (c, top) as well as the estimation of instantaneous hematocrit (c, bottom). (d) Consecutive measurements of RBC flow using the line-scan approach and our pulse-shading method (n = 13 vessels). (e–h) Erythrocyte-associated transients (EATs) in single capillaries. (e) Two RBC fluorescence transients are used as time markers to determine locations of the phosphorescence measurements relative to each RBC. (f) Change in the PO2 near a single RBC. Decays were acquired during 200 s (20 recordings, 10 s each) in eleven capillaries. Each decay is an average of 40,000 gates. (g) PO2 values measured in the plasma and in the close vicinity of RBCs. (h) Consecutive line scans acquired in five capillaries to measure the corresponding distance. Error bars represent s.e.m.
Figure 4
Diffusional shunt of oxygen between arterial and venous compartments in the olfactory nerve layer. (a) PO2 measurements along a venule approaching an arteriole. Left, three-dimensional reconstruction of the two vessels (extracted from a two-photon fluorescence stack of images). Venule is in blue and arteriole in red. Right, seven successive PO2 measurements (three to six acquisitions at each point) in the venule. Scale bars, 40 μm. (b) Diffusional shunt of oxygen is independent of vascular fluctuations. The venule lies in the xy plane. Measurements of PO2 were performed repetitively in five points (2.5 s per point). (c) Venous PO2 as a function of the distance, measured in four animals. (d) Diffusional shunt of oxygen at complete arteriolar-venular crossings. Error bars represent s.e.m.
Figure 5
Functional hyperemia, vascular and neuropil PO2 dynamics in response to odor stimulation. (a) Neuronal and vascular responses during odor stimulation. Inset, glomerulus with boundaries that were outlined by olfactory receptor neuron terminals labeled with Calcium Green-1 dextran, and capillaries labeled with fluorescein-dextran. The presynaptic Ca2+ response was recorded over the rectangle. RBC velocity was obtained from line scans drawn in the capillary segment, indicated by the arrow. Scale bar, 20 μm. (b) RBC and PO2 responses in the same capillary. I.v. injection of PtP-C343 allows detection of the concomitant increases in the RBC flow and PO2 (point measurements of PtP-C343 fluorescence and phosphorescence, respectively) in response to the odorant inhalation (two trials). (c) Mean RBC flow and PO2 responses (nine trials). (d) Summary of vascular responses in five rats. (e) PO2 dynamics in the glomerular neuropil. Left, schematic of the experiment. Middle, three successive measurements of PO2 in neuropil in response to odor stimulation. Right, superposition of presynaptic Ca2+, RBC and PO2 responses in the same glomerulus. Error bars indicate s.e.m.
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