Figure 3. Ratiometric pH imaging of endosomal compartments in meningeal phagocytes using ApHID-dextran sensors.

(A) Experimental strategy. Wild-type mice were implanted with chronic cranial windows and injected with dextrans intraperitoneally (i.p.) 2–3 weeks later. The meninges were continuously imaged by intravital microscopy 15 min after injection, for up to 2h.

(B) Schematic representation of dextran internalization by fluid-phase pinocytosis as typically seen in phagocytic cells such as macrophages. Internalization times for each compartment are based on cell culture studies.

(C) Intravital imaging of meningeal phagocytes (MΦ) in a C57BL/6J mouse injected with fluorescein-Alexa 546-dextrans (70 kDa). Dextrans incorporated into the vasculature, which also appears labeled. Side panels (i-ii) show individual fluorescence channels.

(D) J774A.1 murine macrophages with late endosomes and lysosomes (LE/Lys) labeled with fluorescein-Alexa 546 dextrans (70 kDa), fixed in 0.5% PFA and incubated in pH 5.0 buffer containing membrane-permeant equilibrators at 37 °C. The cells were imaged alongside mice during the same intravital imaging session as a fixed-cell reference for calibration. Side panels (i-ii) show individual fluorescence channels.

(E) Fluorescein/Alexa 546 fluorescence ratios calculated for individual meningeal phagocytes or individual blood vessels (n=1 mouse) and fixed J774A.1 macrophages in pH 5.0 buffer (D).

(F) Representative intravital images of meningeal phagocytes in a C57BL/6J mouse injected i.p. with ApHID-Alexa 546-dextrans (70 kDa). Dextrans incorporated into the vasculature, which also appears labeled. Images were acquired from the same region at various times post-injection (F1-F3). The inset shows individual phagocytes with abundantly labeled acidic vesicles (F3i). Side panels show individual fluorescence channels.

(G) J774A.1 murine macrophages labeled with ApHID-Alexa 546 dextrans (70 kDa), fixed and incubated in pH 5.0 buffer at 37 °C, imaged immediately following intravital imaging as described in (D).

(H) ApHID/Alexa 546 fluorescence ratios calculated for individual meningeal phagocytes and blood vessels at 70–125 min post-i.p. injection time (F2-F3, n=3 mice, shown individually) or for fixed J774A.1 macrophages (ratios per field) in pH 5.0 buffer at 37 °C (G).

(I) ApHID/Alexa 546 ratios measured in fixed macrophages in pH 5.0 buffer (G) were used to generate a ratio-to-pH standard calibration curve, with IC₅₀ corresponding to ApHID's pKa (~5), as previously described¹⁵. One dish was incubated in pH 5.0 buffer for 36 min at 37 °C and imaged thereafter (8 fields acquired) at the end of each technical replicate. Symbols indicate mean ratio per pH point. Error bars (SEM) fit within the symbols. The dotted curve corresponds to the sigmoidal fit.

(J-K) ApHID/Alexa 546 fluorescence ratios for each segmented meningeal phagocyte (MΦ) were translated to pH by interpolation to a ratio-to-pH calibration (as in I) and plotted per mouse (J) or pooled together (K).

In plots (E), (H) and (J), symbols correspond to values for individual meningeal phagocytes, blood vessels or fields containing fixed J774A.1 macrophages. Bars indicate mean ratio or pH ±SD. In (K), dotted lines indicate median and quartiles. Scale bars: 10 μm. Abbreviations: MΦ: meningeal phagocytes; dex: dextran; b.v.: blood vessels; i.p.: intraperitoneally.