Chuyen A et al. (2021), A live-imaging protocol to track cell movement ...

XB-ART-58624

STAR Protoc 2021 Nov 02;24:100928. doi: 10.1016/j.xpro.2021.100928.

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A live-imaging protocol to track cell movement in the Xenopus embryo.

Chuyen A , Daian F , Pasini A , Kodjabachian L .

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Tracking individual cell movement during development is challenging, particularly in tissues subjected to major remodeling. Currently, most live imaging techniques in Xenopus are limited to tissue explants and/or to superficial cells. We describe here a protocol to track immature multiciliated cells (MCCs) moving within the inner epidermal layer of a whole embryo. In addition, we present a data processing protocol to uncouple the movements of individual cells from the coplanar drifts of the tissue in which they are embedded. For complete details on the use and execution of this protocol, please refer to Chuyen et al. (2021).

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Species referenced: Xenopus laevis

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	Figure 1. Embedding of embryos for live imaging(A) Equipment and materials required for experiment: 100 mL glass bottle containing molten 0,8% low melting point agar in 0,1× MBS; box of 24∗40 mm coverslips; a petri dish containing transfected stage 14 embryos with MCCs expressing fluorescent reporter; plastic pipette or flame-polished glass Pasteur pipette, needle-less syringe filled with silicon grease and forceps.(B) Using a 10 mL needle-less syringe filled with silicon grease, delimit a roughly 2cm2 area chamber onto a 24∗40 mm coverslip but do not completely close the chamber.(C) Image of a 2cm2 open silicon chamber onto a 24∗40 mm coverslip.(D) Silicon chamber filled with 3–4 drop of molten agar and 4 embryos.(E) Image of an embryo with field of GFP-positive cells oriented upward.(F) Embedded embryos between two coverslips ready for live imaging.(G) Schematic illustration of embryo embedding between two coverslips. Right panels represent transversal view of flatten embedded embryo, which can be imaged from both sides.
	Figure 2. Illustration of a movie before and after the different steps of stabilizationInitial time point (top) and final time point (bottom) from projection of time-lapse movies (Methods video S1) are shown in original, refocused and stabilized format (refer to Quantification and Statistical Analysis, step 1 to 2). In the original movie, a reference vertex (white arrowhead) is tracked frame by frame through the movie. Using extracted coordinate of the tracked vertex, images are refocused to preserve the original position of that vertex. Next, stabilized images are obtained by using the “Image Stabilizer” Fiji plugin, which implements the Lucas-Kanade algorithm to further stabilize the movie. On the bottom images, the dotted squares demarcate the conserved frame with stabilized position. In the original movie, the tracked vertex has drifted from its initial position. In refocused and stabilized movies, the frame of interest is repositioned to maintain the tracked vertex on its initial position. Note that in refocused and stabilized movies, the MCC tracked in white has been cut by the reframing. Consequently, after reframing only MCCs within the conserved frame can be scored.
	Figure 3. Live imaging of multiciliated cells in Xenopus embryonic epidermis(A) Frames from unstabilized Methods Video S2 showing GFP-labelled MCCs within the epidermal inner layer and mFRP-labelled outer-layer cell junctions. MCCs shows irregular intensity of GFP-labelling, which is due to mosaicism caused by unequal plasmid inheritance. In the field, not all MCCs are labeled and fluorescence intensity increase with time, leading to the detection of additional MCCs during recording. Scale bar is 25 μm.(B) Frames from Methods video S3 showing dynamic actin remodeling of a LifeActGFP-labelled MCC with generation of lamellipodia/filopodia protrusions. Scale bar is 15µm.(C) Live signal of a single MCC from Methods video S4 showing the generation of LifeActGFP-labeled actin rich protrusions stimulated by local enrichment of Arp2/3 actin nucleation factor labelled with eGFP-p41. Scale bar is 15 μm.(D) Frames from stabilized Methods video S5 showing tracked LifeActGFP-positive MCCs dispersing within the inner layer by mutual repulsion. Color dots represent the instant centroid of 4 tracked MCCs and the lines shows the migration path of these MCCs. Events of contact among MCCs are correlated with change in the direction of migration of colliding MCCs and evolution in their instant speed. Scale bar is 25 μm.(E) Graph showing instant speed of MCC n°1 (dark blue) and MCC n°2 (red). After one hour, as MCC n°1 entered in collision with MCC n°3 (green) and MCC n°4 (light blue) its instant speed decreased. At the same time, MCC n°2 separated from MCC n°3 and its instant speed increased.
	Figure 4. Examples of excessive drift where stopping image acquisition is preferable because it may result in non-exploitable data(A) Frames from Methods video S6 showing excessive x,y drift. Several vertices are tracked with color asterisks showing that between time t and t+1, each tracked vertex completely crosses the recorded field.(B) Frames from Methods video S7 with a selected zone of interest on the border of the flattened region of the embryo. The yellow asterisk shows a single tracked vertex illustrating the significant tissue drift and the loss of information in the upper right corner due to embryo curvature.(C) Frames from Methods video S8 illustrating the rapid loss of focus caused by excessive z drift.In all cases, scale bar is 25 μm.
	Figure 5. Examples of manual adjustment in drifting sample during image acquisition(A) Import the sequential images into Fiji (ImageJ) as hyperstacks from ongoing live imaging experiment. Position the hyperstacks on the channel 1, which corresponds to outer-layer cells labeled with mRFP at time point 1 (initial time point) and z 25/25 (the top z on the series).(B) Zoom on image and select a vertex, which will be used as a reference to correct x,y drift over time. Draw an arrow with the arrow tool on this vertex. The arrow will remain in constant position, while the vertex will change position if the tissue drifts.(C) Green double arrowhead illustrates the drift in x and y axes after 9 min of acquisition. To optimize exploitation of the ongoing experiment the drift should be corrected.(D) Illustration of x and y manual adjustment during the time pause between two z-series. The experimenter has used the control joystick to reposition the tracked vertex to its initial position based on the estimated x,y drift.(E) Twenty minutes later, the z drift has caused the signal of outer-layer junctions on the most apical recorded z to not correspond to the surface of the sample.(F) Illustration of z axis manual adjustment during the time pause between two z-series. The experimenter has used the control joystick to reposition the top z to the surface of the sample.
	Figure 6. Image stabilization quality varies with magnification(A–E) Still views of initial time point (left) and final time point (right) from time-lapse movies recorded at 40× (Methods videos S9A and S9B) or 20× magnification (Methods videos S10A and S10B) before (A and C) and after stabilization (B and D). For each image, the red signal corresponds to outer-layer cells labeled with mRFP and the green signal corresponds to MCCs expressing LifeActGFP. The drift between initial and final time points are illustrated with labeled vertices (light blue, dark blue and red). (E) Initial time point (left) and final time point (right) from a cropped section of the movie (Methods video S10C). The crop was extracted from the region boxed in Figure 6D and stabilized using the vertex shown with the white arrowhead as reference.(A′–E’) Dots and lines in light blue, dark blue and red represent the drift of the corresponding tracked vertices over time in movies before (A′ and C′) or after stabilization (B′, D′, and E′). In stabilized movies (B′, D′, and E′) all vertices are not stabilized with the same efficiency, as a consequence of heterogeneity in cellular rearrangement.(F) Dot plot showing the drift, as a distance between the position of vertices at initial and final time points. While image stabilization significantly decreases the drift of most vertices filmed at 40× magnification, the drift is incompletely corrected for vertices filmed at 20× magnification. The quality of the stabilization decreases with the distance to the reference vertex. The drift is significantly corrected in movies cropped to the size of a 40× magnification. For all images, the scale bar is 25 μm. ∗∗∗∗ is p-value <0.0001.

References [+] :

Bostock, An Immobilization Technique for Long-Term Time-Lapse Imaging of Explanted Drosophila Tissues. 2020, Pubmed