Label-free imaging of M1 and M2 macrophage phenotypes in the human dermis in vivo using two-photon excited FLIM
- Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt- Universität zu Berlin, and Berlin Institute of Health, Department of Dermatology, Venerology and Allergology, Germany
- Lomonosov Moscow State University, Faculty of Physics, Russian Federation
Figures
Figure 1
Dermal monocyte skin infiltration and CD68 stained M1 and CD163 stained M2 ΜΦs distribution in excised human skin.
Schematic illustration of monocyte (MO) (green) infiltration into tissues and macrophage (ΜΦ)-polarization into M1 ΜΦs (yellow) via IFN-γ, LPS, and TNF and M2 ΜΦs (blue) via IL-4, IL-13, and IL-33 (a…
Figure 2 with 4 supplements
ΜΦs polarised from PBMC and isolated dermal ΜΦs show distinct TPE-FLIM signatures.
TPE-FLIM τm images (mean fluorescence lifetime τm in the 150–1600 ps range) of monocyte-derived M1-polarised (IFN-γ) ΜΦs (a), monocyte-derived M2-polarised (IL-4) ΜΦs (b), and isolated human dermal …
Figure 2—figure supplement 1
TPE-FLIM visualization of PBMC and histogram of TPE-FLIM parameter.
TPE-FLIM τm images (mean fluorescence lifetime τm in the 150–1600 ps range) of PBMC (a) Scale bar: 10 µm. The distribution of TPE-FLIM parameters τ1, τ2, and τm for PBMC (n=15) (b).
Figure 2—figure supplement 2
In vitro 2D segmentation for ΜΦs from PBMC.
2D segmentation of the τ1(τ2) (a), τ1(a1/a2) (b), τ2(a1/a2) (c), and τ2((a1–a2)/(a1+a2)) (d) TPE-FLIM parameters of the M1 ΜΦs (n=21, orange circles) and M2 ΜΦs (n=27, blue circles) measured in vitro.
Figure 2—figure supplement 3
In vitro segmentation for ΜΦs from dermal tissue.
2D segmentation of the τ1(τ2) (a), τ1(a1/a2) (b), τ2(a1/a2) (c), and τ2((a1–a2)/(a1+a2)) (d) TPE-FLIM parameters of the M1 ΜΦs (n=34, orange circles) and M2 ΜΦs (n=28, blue circles) measured in …
Figure 2—figure supplement 4
Time stability of TPE-FLIM parameters for ΜΦs isolated from periocular skin in vitro.
TPE-FLIM images of M1 and M2 ΜΦs in vitro (shown in dotted ovals) showing mean fluorescence lifetime τm in color gradient from 150 to 1600 ps recorded with the time interval of 60 min. Scale bar: 15 …
Figure 3 with 2 supplements
M1 and M2 ΜΦs ex vivo verified using TPE-FLIM parameters and immunohistochemistry-based bright field microscopy.
Side by side comparison of TPE-FLIM τm images (mean fluorescence lifetime τm in the 150–1600 ps range), which were measured label-free and then stained with CD68-antibody for M1 ΜΦs (a), and …
Figure 3—figure supplement 1
Ex vivo segmentation for ΜΦs from skin biopsies.
2D segmentation of the τ1(τ2) (a), τ1(a1/a2) (b), τ2(a1/a2) (c), and τ2((a1–a2)/(a1+a2)) (d) TPE-FLIM parameters of the M1 ΜΦs (n=8, orange circles) and M2 ΜΦs (n=12, blue circles) measured ex vivo.
Figure 3—figure supplement 2
Mast cell-specific staining with tryptase ex vivo – negative control.
Side by side comparison of TPE-FLIM image (average lifetime τm in the 300–1300 ps range) (a) and corresponding light microscopic image after staining of human skin biopsy cryo-sections for MCs (b). …
Figure 4 with 6 supplements
ΜΦs are visualized and categorised by TPE-FLIM signatures in vivo.
TPE-FLIM in vivo images of potential perivascular flat spindle shaped M1 ΜΦ (a), of suspected slightly dendritic M1 ΜΦ in the depth 90 µm (b) large intervascular M1 ΜΦ with membrane extensions (c) …
Figure 4—figure supplement 1
In vivo segmentation for ΜΦs in human skin.
2D segmentation of the τ1(τ2) (a), τ1(a1/a2) (b), τ2(a1/a2) (c), and τ2((a1–a2)/(a1+a2)) (d) TPE-FLIM parameters of the M1 ΜΦs (orange circles) and M2 ΜΦs (blue circles) measured in vivo. Cells …
Figure 4—figure supplement 2
Time stability of TPE-FLIM parameter for M1 ΜΦ in vivo.
TPE-FLIM images recorded in 5-min interval for 30 min of M1 ΜΦ measured in vivo in human papillary dermis at the depth 90 µm showing mean fluorescence lifetime τm in color gradient from 150 to 1600 …
Figure 4—figure supplement 3
Time stability of TPE-FLIM parameter for M2 ΜΦ in vivo.
TPE-FLIM images recorded in 5-min interval for 30 min of M2 ΜΦ measured in vivo in human papillary dermis at the depth 96 µm showing mean fluorescence lifetime τm in colour gradient from 150 to 1600 …
Figure 4—figure supplement 4
TPE-FLIM allows for visualization of potentially phagocytosing M1 ΜΦs in vivo.
Merged TPE-AF (green) and SHG (red) image of possibly activated (phagocytosing) M1 ΜΦ measured in vivo in human papillary dermis at the depth 105 µm (a) and corresponding TPE-FLIM image (b), showing …
Figure 4—figure supplement 5
Decision tree model.
Decision tree model for classification of M1 and M2 ΜΦs with the parameters entropy impurity, minimal samples for split is 2, maximum depth of the tree is 9, and the samples had equal weight. The …
Figure 4—figure supplement 6
ROC curves of decision tree models.
ROC curves of false positive to false negative rates for classification of ΜΦs versus other dermal cells (a) and for classification of M1 ΜΦs (orange line) versus M2 ΜΦs and other dermal cells and …
Appendix 1—figure 1
The dependence of fluorescence decay parameters for individual macrophages (n=110) on the integral fluorescence intensity (area under the fluorescence decay curve, upper row).
As can be seen, the fluorescence decay parameters were independent on the intensity. To further confirm the absence of artefacts connected with parameters dependence on the FLIM data quality and …
Appendix 1—figure 2
The dependence of fluorescence decay curves parameters on χ2.
Each point corresponds to an individual macrophage cell (n=110). The dependence of χ2 on fluorescence intensity (both integral intensity per pixel and amplitude of the fluorescence decay curve, …
Appendix 1—figure 3
Distribution of the χ² for the bi-exponential (left) model and three-exponential (right) decay models.
Author response image 1
Fluorescence decay curves for individual macrophages, from which the values of τ1 < 300 ps (left) and τ1 > 300 ps (right) were obtained using the biexponential decay model.
The black curve corresponds to IRF of the setup. The curves were normalized to the maximum.
Author response image 2
Correlations between the phasor plot parameters (c and s) and fluorescence decay parameters obtained from the biexponential fitting.
Each point corresponds to an individual macrophage.
Tables
Table 1
TPE-FLIM parameters for investigated dermal and epidermal cells.
TPE-FLIM parameters τ1, τ2, τm, a1/a2 and TPE-AF intensity of monocyte-derived M1 and M2 ΜΦs; dermal M1 and M2 ΜΦs isolated from the skin measured in vitro; M1 (CD68) and M2 (CD163) ΜΦs measured ex …
| Number of cells | τm in ps | τ1in ps | τ2in ps | a1/a2 | TPE-AF intensity, photons /mW | |||
|---|---|---|---|---|---|---|---|---|
| in vitro | Monocyte-derived M1-polarised ΜΦs | 21 | 479±106 | 163±50 | 1,209±161 | 2.4±0.6 | 600±100 | |
| in vitro | Monocyte-derived M2-polarised ΜΦs | 27 | 1,185±170 | 417±134 | 2,305±194 | 2.3±0.5 | 500±100 | |
| in vitro | M1 isolated dermal ΜΦs | 34 | 461±175 | 225±84 | 1,289±278 | 4.8±3.4 | 3,000±500 | |
| in vitro | M2 isolated dermal ΜΦs | 28 | 1,281±155 | 807±250 | 2,352±229 | 2.2±1.1 | 800±200 | |
| ex vivo | M1 ΜΦs (CD68) | 8 | 458±50 | 190±38 | 1,504±133 | 4.1±0.7 | 3,000±500 | |
| ex vivo | M2 ΜΦs (CD163) | 12 | 1,369±201 | 498±129 | 2,267±155 | 1.1±0.4 | 700±300 | |
| in vivo | M1 ΜΦs | 35 | 477±105 | 196±40 | 1,698±172 | 5.0±2.8 | 686±165 | |
| in vivo | Phagocytosing M1 ΜΦs | 2 | 195±44 | 105±10 | 1,272±89 | 14.7±4.5 | 1,100±150 | |
| in vivo | M2 ΜΦs | 25 | 1,407±60 | 442±54 | 2,458±90 | 1.2±0.2 | 360±155 | |
| in vitro | PBMC-derived monocytes | 15 | 989±111 | 491±130 | 2,025±301 | 1.8±0.5 | 700±130 | |
| in vitro | Resting mast cells | 43 | 1,248±287 | 533±266 | 2,289±317 | 1.5±0.5 | 1,300±400 | |
| in vitro | Activated mast cells | 13 | 862±268 | 288±130 | 1,920±287 | 2.5±2.0 | 900±200 | |
| in vitro | Dendritic cells | 14 | 1,265±180 | 434±188 | 2,578±328 | 1.6±0.2 | 538±258 | |
| in vitro | Fibroblasts | 6 | 921±81 | 429±51 | 1,983±137 | 0.5±0.1 | 469±137 | |
| in vitro | Neutrophils | 21 | 1,074±109 | 714±250 | 1,795±600 | 1.5±0.5 | 500±115 | |
