Parameters used for individual cell analysis.

<p>A: Edge detection of two CX₃CR1+ pulmonary cells and their roundness coefficient. Scale bar  = 10 µm. B: The relevant parameters used in this work are: i) the Mean Roundness Coefficient (MRC), calculated for each cell by meaning Instantaneous Roundness Coefficient (IRC) at each consecutive observable time; ii) the Maximal Distance (MD) of a cell (red arrow) is the longest distance covered from the first position; iii) the Meandering Index (MI) is the final distance from the first position <i>D_n</i> divided by the total length covered.</p

Velocity of CX₃CR1-GFP positive pulmonary Dendritic-shaped and Round-shaped cells.

<p>A: Dendritic-shaped cells and B: Round-shaped cells at an early stage (average values from 1h30 to 2h30 post injection, closed symbols) and a late stage (average values from 5h to 6h post injection, open symbols) after injection of PBS (rounds) or LPS (squares). Three mice in each group, one symbol by cell. * for p<0.05; ** for p<0.01; *** for p<0.0001; ns for not significant.</p

Phenotype of CX₃CR1-GFP cell subsets in the lung.

<p>A: Total lung cells of CX₃CR1^+/gfp mice were gated on CX₃CR1 and analyzed for NK1.1, CD3e, CD11c, and CD11b expressions. B: Autofluorescence, CD80 and MHCII expressions on gate G1, G2 and G3 of panel A. Black histogram, isotype control; grey histogram, positive staining. C: Total lung cells were pre-gated on CD11c+ low autofluorecent cells and analyzed for the expression of CD11b and CD103. The expression of CX₃CR1 is shown on the left panel for gate G4 (CD11b⁻CD103⁺ DCs, grey histogram) and for gate G5 (CD11b⁺CD103⁻ DCs, black line). D: Total cells were pre-gated on CD45 cells and analyzed for the expression of F4/80 and CD11c. Expression of CX₃CR1 and CD11b is shown on the left panels for gate G6 (CD11c^lowF4/80^high), G7 (CD11c^highF4/80^high) and G8 (CD11c^highF4/80^low).Data from flow cytometry, performed on one CX₃CR1^+/gfp mouse lung harvested 30 minutes after intratracheal PBS injection. Data are representative of two distinct experiments.</p

Discrimination of two CX₃CR1-GFP cell subsets using flow cytometry or roundness.

<p>A: Expression of CX₃CR1 <i>vs</i> CD45 receptors in pulmonary cells and CD11c <i>vs</i> CD11b by CX₃CR1^high cells. Data from flow cytometry, performed on one CX₃CR1^+/gfp mouse lung harvested 30 minutes after intratracheal PBS injection. Data representative of two distinct experiments. B: Frequency distribution of Mean Roundness Coefficients (MRC) over 1h of CX₃CR1+ pulmonary cells in lung slices harvested from 6 mice, 1h30 (3 mice) and 5h (3 mice) after PBS injection. N = 406. Cells are followed for a mean time of 32 minutes, corresponding to 16 frames. Dashed line: sum of two Gaussian fitting the histogram. R² = 0.89.</p

Schematic representation of the drift correction strategy.

<p>A: Maximum intensity projection of a lung slice z-stack. Pulmonary CX₃CR1-GFP cells (green) and alveolar collagen mesh detected by collection of SHG signal (gray). Two-photon excitation wavelength  = 896 nm. B: Dashed red squares show the optic field imaged by the microscope (CX₃CR1-GFP in green and SHG in grey). The realignment phase consists in calculating the tissue drift using the maximization of SHG signal cross-correlation.</p

Maximal Distance of CX₃CR1-GFP positive pulmonary Dendritic-shaped and Round-shaped cells.

<p>A: Dendritic-shaped cells and B: Round-shaped cells at an early stage (average values from 1h30 to 2h30 post injection, closed symbols) and a late stage (average values from 5h to 6h post injection, open symbols) after injection of PBS (rounds) or LPS (squares). Three mice in each group, one symbol by cell. C, D: Overlay of Round-shaped cell tracks after late PBS (C) and LPS injection (D), after aligning their first coordinates. One color by track. Values of black circle radii in µm, equal to average cell Maximal Distance, are indicated ± standard deviation. * for p<0.05; ** for p<0.01; *** for p<0.0001; ns for not significant.</p