A new platform for multiplexed longterm imaging of cell behaviour during regeneration and development.

Being able to image cells using microscopy is central to many biological research projects. It is often useful to follow living cells to understand their behaviour. Research tools such as fluorescent proteins are available that let one study the minute details of cells in realtime. However, imaging cells within a dense tissue is particularly difficult as the cells above and below interfere with image capture. To partially overcome this issue microscopes that illuminate samples from the side with a "light sheet" have been developed that reduce interference. Further technology is needed to track cells over long periods such as days. Tissues have special environmental needs in order to stay healthy for long periods, and using too much light on the sample can also be hazardous. In addition, if the cells move we need the microscope to follow them automatically. Our researchers are requesting the purchase of an advanced microscope that overcomes these hurdles to enable successful longterm imaging of dense tissues.

Our project involves the purchase of an advanced lightsheet microscope LSM called the TrueLive3D. This LSM is ideally suited for long-term imaging of larger, more complex samples. Originally developed for imaging of organoids at the EMBL in Heidelberg, this system uses a FEP trough to hold the samples in place whilst imaging from below. The trough-based format has many advantages. Samples are laid in the bottom of the trough and are held in place by gravity and the sides of the trough. This cradles the sample while allowing it to grow. Drift is slow because the small size and shape of the trough minimises currents around the sample. Another important feature of this LSM is real-time phase drift correction. Drift correction compares recent images of the sample to determine any drift in X, Y and Z and moves the samples during image capture to correct for movement. This not only ensures that the sample does not drift out of the field of view, but also allows the researcher to use greater magnification. Multiplexing is another asset: the standard trough is 40mm long allowing for many samples to be imaged at once. Chemical treatment in small volumes (0.1 to 1.0ml) is possible because the trough is isolated from the water in the surrounding lens chamber. This isolation also facilitates growth in media/matrix for culturing organoids and explants. Troughs are also available with individual wells which increases experimental flexibility (e.g. a 4 well trough with each well holding multiple samples enabling four chemical treatments to be analysed in parallel). These attributes will have a transformative effect on our ability to use imaging in our experimental protocols and to design new research proposals.