Table 1.
Experimental conditions of live imaging of microglia in vivo.
| Animal | Age | Skull | Anesthesia |
Region (depth from the surface) |
Cellular visualization | Microscopy | Objective lens |
Resolution (x, y) z stack |
Interval time Total time |
References |
|---|---|---|---|---|---|---|---|---|---|---|
| CX3CR1-GFP+/− mice | 2 mo | Thinned | Isoflurane | Cortex | GFP (microglia) | Two-photon | 25x (NA = 1.05) | – 1 μm step |
1.5 min 13.5 min |
Abiega et al. (11) |
| CX3CR1-GFP+/− mice | 8–12 mo | Open | Isoflurane | Cortex | GFP (microglia) | Two-photon | 20x (NA = 1.0) | 317 × 317 μm 50–80 μm with 1 μm step |
– 22 days | Askew et al. (12) |
| CX3CR1-GFP+/− mice | 8–10 wo | Thinned | – | Cortex (100–150 μm) | GFP (microglia) mCherry (axonal bouton) |
Two-photon | 25x (NA = 1.10) | 1,024 × 1,024 pixel (0.1269 μm/pixel) 1 μm step |
1 min 3 h |
Badimon et al. (13) |
| C57BL/6 mice | 2–6 mo | Thinned | Isoflurane | Somatosensory cortex (~330 μm) | Twitch-2B (microglia) | Confocal | 20x (NA = 1.0), 40x (NA = 0.8) | 0.15–0.31 μm/pixel | 1 frame/s, 10 min or 0.13 frame/s, 30–120 s | Brawek et al. (14) |
| pU.1::Gal4-UAS::TagRFP, mpeg1::Gal4-UAS::Kaede, nbt::DlexPR::NTR-mCherry, and slc7a7::Kaede (zebrafish) | 2–3 dpf | – | 0.01% tricaine | RFP, Kaede (microglia) mCherry (neuron) |
Confocal | 20x (NA = 0.4), 10x (NA = 0.25) | – 30–40 planes with 1.5–2 μm step |
– | Casano et al. (15) | |
| C57BL/6 mice | P4-19 | Open | Isoflurane | Cortex (300–450 μm) | EGFP (neuron) | Two-photon | 40× | 78 × 79 μm 20 sections with 1 μm step |
– ~2 h |
Cruz-Martin et al. (16) |
| CX3CR1-GFP+/−;Thy1-YFP+/− mice | – | Thinned | Ketamine and xylazine | Cortex (~300 μm) | GFP (microglia) YFP (neuron) SR101 (astrocyte) Hoechst (nuclei) |
Two-photon | 20x (NA = 1.0) | 1,024 × 1,024 pixel (0.204 μm/pixel) 34 planes with 3 μm step |
2–3 h – |
Damisah et al. (17) |
| CX3CR1-GFP+/− mice | – | Thinned | Ketamine and xylazine | Cortex (~200 μm) | GFP (microglia) | Two-photon | 40x (NA = 0.8), 60x (NA = 0.9 or 1.0) | – 0.75–2 μm step |
– | Davalos et al. (2) |
| CX3CR1-GFP+/−;Thy1-YFP+/− mice | – | Thinned | – | Cortex | GFP (microglia) YFP (neuron) Alexa594 (fibrinogen) |
Multi-photon | 10x (NA = 0.4), 40x (NA = 0.8) | – 1.0–1.5 or 3–4 μm step |
120–240 s 30–90 min |
Davalos et al. (18) |
| CX3CR1-GFP+/− mice | – | Open | Mixture of ketamine, xylazine and acepromazine | Spinal cord | GFP (microglia) rhodamine dextran (vessel) |
Two-photon | – | – | – | Davalos et al. (19) |
| CX3CR1-GFP+/− mice | P40-130 | Open | Pentobarbital sodium and methohexital sodium | Spinal cord | GFP (microglia) | Two-photon | 40x (NA = 0.75), 20x (NA = 1.0) | 256 × 256 or 1,024 × 1,024 pixel (0.24–0.72 μm/pixel) 16–24 planes with 1.5–2.0 μm step |
1–2 min – |
Dibaj et al. (20) |
| WT mice CX3CR1-GFP+/− mice |
2–4 mo | Thinned | Isoflurane | Cortex | GFP, OGB-1, Fluo-4, Isolectin B4 conjugated to Alexa Fluor 594 (microglia) | Two-photon | 40x (NA = 0.8), 60x (NA = 1.0) | – 10 μm with 1 μm step |
– | Eichhoff et al. (21) |
| CD11b-CreERT2;R26-tdTomato;APPPS1 mice | 4, 10 mo | Open | Isoflurane | Cortex (200–250 μm) | tdTomato (microglia) | Two-photon | 25x (NA = 0.95) | 0.27 μm/pixel or 1.49 μm/pixel – |
Biweekly or monthly 1.5 years |
Füger et al. (22) |
| TH-tdTomato+/−; CX3CR1-GFP+/− mice | P56 | Thinned | Isoflurane | Olfactory bulb | GFP (microglia) TH neuron (mCherry) |
Two-photon | 20x (NA = 1.0) | 0.09 μm/pixel or 0.99 μm/pixel – |
30 s 10 min |
Grier et al. (23) |
| Thy1-YFP+/− mice CX3CR1-EGFP+/− mice |
– | Thinned | Ketamine and xylazine | Cortex (~100 μm for neuron and ~200 μm for microglia) | YFP (neuron) EGFP (microglia) |
Two-photon | 60x (NA = 1.1) | 512 × 512 pixel (66.7 × 66.7 μm) 0.75 μm step |
– ~2 h |
Grutzendler et al. (24) |
| Thy1-YFP+/− mice (H-line) | 6–7 mo | Open | Ketamine and xylazine | Hippocampus (50 μm below the pyramidal cell layer) | YFP (neuron) | Two-photon | 16x (NA = 0.8) | 100 × 100 μm (0.09 μm/pixel) 60 μm with 1 μm step |
4 d interval – |
Gu et al. (25) |
| CX3CR1-GFP+/− mice | – | Thinned | – | Cortex | GFP (microglia) | Two-photon | - | – 15 planes with 2 μm step |
4 min 40 min |
Haynes et al. (26) |
| mpeg1:GFP (zebrafish) | 4 dpf | – | 0.01% MS-222 | Brain (100–150 μm) trunk(80–120 μm) |
GFP (microglia) PI (dead cell) |
Confocal | - | – 3.6–6 μm step |
6 min – |
Herzog et al. (27) |
| CX3CR1-GFP+/−;Cnp-mEGFP+/−;Plp-DsRed+/− mice | P30-1,100 | Thinned or open | – | Somatosensory cortex (~75 μm) | GFP (microglia) mEGFP (myelin) DsRed (oligodendrocyte) |
Confocal or two-photon | 20x (NA = 1.0) | – | – | Hill et al. (28) |
| CX3CR1-GFP+/− mice | 6–10 wo | Thinned | Awake or mixture of ketamine and xylazine or pentobarbital | Somatosensory cortex (50–150 μm) | GFP (microglia) | Two-photon | 20x (NA = 0.95) | 521 × 521 pixel (0.38 μm/pixel) 26–37 planes with 1 μm step |
30 s 15–20 min |
Hristovska et al. (29) |
| NSG-CCR2-RFP+/−;CX3CR1-GFP+/− mice | 8–12 wo | – | Isoflurane | Frontal cortex | GFP (microglia) RFP (macrophage) BFP (tumor) |
Two-photon | 20x (NA = 0.95) | 1,024 × 1,024 pixel (0.584 μm/pixel) 7 planes with 1 μm step |
– | Hutter et al. (30) |
| Thy1-GFP+/− mice (M-line) | – | Open or thinned | Ketamine and xylazine | Somatosensory cortex (~100 μm) | GFP (neuron) | Two-photon | 25x (NA = 1.05) | 78 × 78 μm (0.15 μm/pixel) 0.75 or 0.5 μm step |
90 min – |
Isshiki and Okabe (31) |
| Tg(Apo-E:eGFP) (zebrafish) | 5–8 dpf | – | Awake | – | GFP (microglia) OGB-AM (Ca2+) |
Confocal or two-photon | 40x (NA = 0.80) | – 1 μm step |
2–4 s or 1.5–5 min |
Li et al. (32) |
| CX3CR1-GFP+/− mice | 2–3 mo | Open | Awake or isoflurane or ketamine and xylazine | Cortex | GFP (microglia) | Two-photon | 40x | 512 × 512 pixel (0.35 μm/pixel) 8 planes with 2 μm step |
1 min – |
Liu et al. (33) |
| Iba1-EGFP+/− mice | P8-10 | Open | Urethane and atropine or awake | Cortex (45–250 μm) | GFP (microglia) tdTomato (neuron) GCaMP6m (Ca2+) |
Two-photon | 25x (NA = 1.05) | 512 × 512 pixel (0.099 μm/pixel) 0.5 μm step |
5 min, 30 min-2 h or 1.6 s-1 min, 27 min or 8 Hz, 30 min | Miyamoto et al. (34) |
| Tg(mpeg1:GAL4, UAS:mCherry); rwTg(isl1:GFP); Tg(-3.5ubb:secAnnexin V-mVenus) (zebrafish) | 2–5 dpf | – | 0.01% tricaine | Spinal cord | mCherry (microglia) GFP (neuron) mVenus (AnnexinV) |
Confocal | 10x (NA = 0.3), 40x (NA = 0.8), 63x (NA = 0.9) | – 10–15 planes with 1–2 μm step |
3–8 min | Morsch et al. (35) |
| CX3CR1-GFP+/− mice | 1.5–15 mo | Thinned | Isoflurane | Cortex (75 μm) | GFP (microglia) | Two-photon | – | – 15–25 planes with 1–2 μm step |
20–45 s Several hours |
Nimmerjahn et al. (1) |
| CX3CR1-GFP+/− mice | 2–4, 9–11, 18–21 mo | Thinned | Isoflurane | Cortex (80–100 μm) | GFP (microglia) Oregon Green 488 (Ca2+) |
Two-photon | 40x (NA = 0.80) | – | 4 frames/s or 10 frames/s or 30 s, 20 min | Olmedillas Del Moral et al. (36) |
| CX3CR1CreER/+: Thy1 YFP-H mice | P19-34 | Thinned | Ketamine and xylazine | Motor cortex (~100 μm) | YFP (neuron) | Two-photon | 60x (NA = 1.1) | – | 4 d – |
Parkhurst et al. (37) |
| ApoE-GFP;NBT-DsRed (zebrafish) | 3 dpf | – | 0.01% tricaine | – | GFP (microglia) DsRed (neuron) LysoTracker DND-9 (lysosome) |
Confocal | 40x (NA = 1.2) | – 4 stacks spanning 10 μm |
– | Peri et al. (38) |
| PC::G5-tdTomato mice | 2–4 mo | Thinned | Isoflurane | Visual or somatosensory cortex (~100 μm) | tdTomato (microglia) GCaMP5G (Ca2+) |
Two-photon | 16x (NA = 0.8) | 512 × 512 or 1,024 × 1,024 pixel | 0.125 frames/s or 0.5 Hz – |
Pozner et al. (39) |
| B6.Cg-Tg(Thy1-YFP)HJrs/J mice (#003782, JaxLab) | 4–10 wo | Open | Isoflurane and ketamine | Somatosensory cortex | YFP (neuron) | Two-photon | 25x (NA = 1.05) | 166.7 × 166.7 μm (512 × 512 or 800 × 800 pixel) 30–70 μm with 1 μm step |
24 h – |
Pryazhnikov et al. (40) |
| CX3CR1-GFP+/−;Thy1-YFP+/− mice | 3–17 wo | Open | Awake or dexmedetomidine and midazolam | Cortex | GFP (microglia) YFP (neuron) |
Two-photon | 20x (NA = 0.95) | – 1 μm step |
5 min 1 h |
Stowell et al. (41) |
| CX3CR1-GFP+/− mice | 3–4 mo | Open | Awake or isoflurane or ketamine | Cortex (100–150 μm) | GFP (microglia) | Two-photon | 20x (NA = 1.00) | 512 × 512 pixel (0.77 μm/pixel) 6 planes with 2 μm step |
20 s 33 min |
Sun et al. (42) |
| CX3CR1-GFP+/− mice, CX3CR1CreER-eYFP mice, and Rosa-CAG-LSL-eYFP-WPRE mice | 2–5 mo | Open | Isoflurane | Somatosensory cortex (50–100 μm) | GFP, YFP (microglia) Evans lue,Rhodamine-dextran (vessel) |
Multi-photon | 40x (NA = 0.8) | 800 × 800 pixel (0.18 μm/pixel) – |
4 min 40–60 min |
Taylor et al. (43) |
| CX3CR1-GFP+/−;Thy1-YFP+/− mice | P28-39 | Thinned | – | Primary visual cortex (50 μm) | GFP (microglia) YFP (neuron) |
Two-photon | 20x (NA = 0.95) | – 1 μm step |
5 min 30 min-2 h |
Tremblay et al. (44) |
| Rosa26-CAG-LSL-GCaMP6s mice, Rosa26-CAG-LSL-Lck-GCaMP6f mice, and CX3CR1CreER−eYFP mice | 3–5 mo | Open | Awake | Somatosensory cortex (55–80 μm) | GCaMP6s, GCaMP6f (Ca2+) GFP (microglia) |
Two-photon | 16x (NA = 0.8) | 512 × 512 pixel (300 × 300 μm) | 1 s – |
Umpierre et al. (45) |
| Iba1-EGFP+/−;Thy1-GFP+/− mice | 6–10 wo | Thinned | Ketamine and xylazine | Cortex (100–250 μm) | GFP (microglia) YFP (neuron) |
Two-photon | 60x (NA = 1.1) | – 40–50 planes with 0.5 μm step |
0.3–1.0 s – |
Wake et al. (3) |
| Thy1-YFP+/− mice (H-line) |
2–7 mo | Open or thinned | – | Barrel cortex (0–100 μm) | YFP (neuron) | Two-photon | 60x (NA = 0.9) | – | – | Xu et al. (5) |
| Tg(-2.8elavl3:eGFP; coro1a:DsRedx) (zebrafish) | 1–5 dpf | – | Awake or 0.01% tricaine | – | DsRed-Express (microglia) GFP (neuron) |
Confocal | 20x | – 40–50 planes with 3 μm step |
3–5 min – |
Xu et al. (53) |
| Thy1-YFP+/− mice (H-line) CX3CR1-EGFP+/− mice |
– | Thinned | Ketamine and xylazine | Cortex (~100 μm for neuron and ~200 μm for microglia) | YFP (neuron) EGFP (microglia) |
Two-photon | 60x (NA = 1.1) | 512 × 512 pixel (66.7 × 66.7 μm) 0.75 μm step |
– ~2 h |
Yang et al. (46) |
| Thy1-YFP+/− mice Thy1-GFP+/− mice |
1 mo | Thinned | Ketamine and xylazine | Motor cortex (100–200 μm) | YFP or GFP (neuron) | Two-photon | 60x (NA = 1.1) | 70 × 70 μm (512 × 512 pixels) 9 planes with 0.7 μm step |
– | Yu et al. (47) |
mo, month-old; dpf, days post-fertilization; wo, week-old; P, post-natal day; –, not available.