Proteins and nucleic acids undergo countless encounters in the cell. While some interactions exhibit well-defined stoichiometric association, others amount to assembly of the biomolecules into higher order complexes. One prominent mechanism of the assembly is LLPS which occurs when the local concentration of biomolecules reaches a critical point, termed saturation concentration (Csat), and demixes to form liquid-like condensates.

The studies exploring phase separated condensates such as P granules [1] spurred more recent investigations on the formation-function relationship of biomolecular condensates. Using genetics and proteomics data, researchers sought to catalog the protein composition of diverse cellular condensates including stress granules and P-bodies [2, 3, 4, 5]. These studies identified proteins that are necessary for the formation of cellular condensates and which undergo phase separation in vitro. Many of these proteins are highly disordered RNA binding proteins (RBPs) which share similar, yet diverse motifs and domains that promote phase separation mediated by protein-protein interaction. These include sticker-like domains such as RNA recognition motifs, arginine-glycine-rich regions, and spacer-like regions such as low complexity domains (LCDs) which promote LLPS by forming reversible, multivalent interactions [6, 7, 8, 9].

Recent advances in microscopy have enabled the measurement of the formation, localization, and material properties of ribonucleoprotein (RNP) condensates. Most tools used to analyze condensates are solution-based in which signals are ensemble averages. To overcome this limitation, the single molecule methods have been used to explore phase separation dynamics with molecular precision. In addition, atomic force, electron, and super-resolution fluorescence microscopy have provided the ability to study condensates with sub-diffraction limited resolution.

Here we will outline recent advances in microscopy and spectroscopy-based methods that provide the means to investigate biomolecular condensates with high spatiotemporal resolution. We will discuss various cutting-edge techniques that pave new paths toward investigating condensate formation in vitro and in cells. These approaches, combined with pre-existing, well-established biochemical and in vitro analyses provide unique and comprehensive mechanistic insights into condensate formation, dynamics, and function.