Visual discrimination of membrane domains in live cells by widefield microscopy

Membrane dynamics is a fast-evolving field with the many new methods and probes being developed each year affording ever increased insights into how membranes behave in the laboratory. Typically, these developments are first tested in model membranes using high-cost, bespoke microscopes which often employ confocal and two-photon systems and which give little consideration to preservation of cellular integrity and homeostasis during experiments. This chapter addresses the clear need to rapidly apply and deploy this work into mainstream biological laboratories by development of economical, four-dimensional imaging on user-friendly low-cost systems using widefield optics and simultaneous capture of multiple fluorescent markers. Such systems are enabling biologists to consider the coordinated processes triggered from signalling platforms during cellular interaction with the environment. In this chapter, we describe the progress made to date and in particular we focus on the Laurdan family of fluorescent probes, which are being used to image whole cells and tissues using widefield epifluorescence microscopy and which can be usefully combined with simultaneous capture at longer wavelengths (yellow through far red) for imaging of cell morphology or for following expressed markers such as fluorescent adaptor proteins.