These enable the live monitoring of gene expression and protein localization in vivo, and in real time. The traditional approach of collecting “static” images of fixed or post mortem cells and tissues provides a snapshot view of events at a single fixed point in time. However, this inherently overlooks the dynamic aspects of the biology being examined. In contrast, live cell imaging enables the visualization of temporal changes in living specimens and can reveal novel aspects of the biology that may not otherwise have been appreciated. Additionally, the datasets generated from time-lapse imaging are information rich and can be interrogated quantitatively to enable measurement of cellular,

subcellular and tissue dynamic events as a function of time (reviewed in [37]). Although these approaches are leading to exciting discoveries find protocol that are advancing our understanding of biological systems, there are several limitations that need to be acknowledged. Firstly, the use of any fluorescent probe has the potential to perturb or alter the biology being examined and this must always be taken into account when interpreting live imaging data. For example, fusion of GFP sequences, which are approximately 27 kDa in size, with the protein of interest may disrupt the normal function of the protein. Therefore, validation studies are needed

to make sure that the fusion protein still functions similarly to the wild type form. It is also advantageous to confirm findings with more than one type of imaging probe if possible. For example, a GFP fusion protein can be used for in Selleck AZD6244 vivo localization of a specific protein and key data can be confirmed using a fluorescence-conjugated antibody against the same protein. When developing live cell imaging protocols, there is always a compromise between obtaining a high enough signal-to-noise ratio to enable quantitative measurements and to obtain sufficient image resolution, while at the same time avoiding phototoxic effects to the cells (reviewed in [36] and [38]). Therefore, to ensure cell

viability, the researcher may have to accept a lower image quality and resolution than would be acceptable for equivalent images of fixed specimens. Light microscopy based live many cell imaging approaches that use widefield or confocal microscopy are also limited by issues such as signal attenuation with depth of penetration into the tissue, as mentioned earlier in this review. For a more extensive discussion of the advantages and limitations of live cell imaging methods in relation to imaging of bone cells, please refer to Dallas and Veno 2012 [36]. Technologies such as multiphoton fluorescence microscopy can increase the depth of tissue penetration for live cell imaging applications and reduce phototoxicity by using a longer wavelength light to excite the fluorophores.