Whitington Lab ~ Time Lapse Microscopy

Time-lapse microscopy for observation of axon growth

We can reconstruct the pattern of growth by an axon by examining its morphology in embryos fixed at successive developmental stages. However the growth cone is a highly dynamic structure and many questions about the growth process can only be answered by examining axons "in the act" using time-lapse microscopy. We have previously developed techniques for studying the dynamics of axon growth from neurons in the CNS of the Drosophila embryo (Murray et al., 1998).

To stain neurons for time-lapse microscopy, we either inject them with DiI or drive expression of GFP in them. As both DiI and GFP are non-toxic to the neurons, we can follow the growth of neurons labelled with these dyes for several hours. Images are collected at short intervals (30s or 1min) using an intensified video-camera and stored on a computer hard disk for replaying as a movie.

This movie shows axon growth from a DiI filled RP2 neuron within the CNS. The axon initially grows anteriorly, then turns laterally into the ISN. Inter-frame interval is 1 minute. Movie by Michael Murray.

In this movie a subset of central axons, including the motorneuron aCC, express tau-GFP (green), while the RP2 neuron has been injected with DiI (orange). This method reveals the close association between the aCC axon and RP2 filopodia as the growth cone of the latter neuron turns into the ISN. Inter-frame interval is 3 minutes. Movie by Michael Murray.

We are currently working to adapt these methods for the study of axon growth from sensory neurons. We also plan to express GFP-tagged cytoskeletal molecules (e.g. F-actin) in the sensory neurons to study how they are redistributed within their axons as they turn through specific choice points.