Advanced optical imaging in living embryos

Developmental biology investigations have evolved from static studies of embryo anatomy and into dynamic studies of the genetic and cellular mechanisms responsible for shaping the embryo anatomy. With the advancement of fluorescent protein fusions, the ability to visualize and comprehend how thousands to millions of cells interact with one another to form tissues and organs in three dimensions (xyz) over time (t) is just beginning to be realized and exploited. In this review, we explore recent advances utilizing confocal and multi-photon time-lapse microscopy to capture gene expression, cell behavior, and embryo development. From choosing the appropriate fluorophore, to labeling strategy, to experimental set-up, and data pipeline handling, this review covers the various aspects related to acquiring and analyzing multi-dimensional data sets. These innovative techniques in multi-dimensional imaging and analysis can be applied across a number of fields in time and space including protein dynamics to cell biology to morphogenesis

Structure–function relationships in fluorescent marker proteins of the green fluorescent protein family

GFP-like proteins, originally cloned from marine animals, are genetically encoded fluorescence markers that have become indispensable tools for the life sciences. The search for GFP-like proteins with novel and improved properties is ongoing, driven by the persistent need for advanced and specialized fluorescence labels for cellular imaging. The 3D structures of these proteins are overall similar. However, considerable variations have been found in the covalent structures and the stereochemistry of the chromophore, which govern essential optical properties such as the absorption/emission wavelengths. A detailed understanding of the structure and dynamics of GFP-like proteins greatly aids in the rational engineering of advanced fluorescence marker proteins. In this chapter, we summarize the present knowledge of the structural diversity of GFP-like proteins and discuss how structure and dynamics govern their optical properties