Nuclear organelles are viscous droplets, created by concentration-dependent condensation and liquid–liquid phase separation of soluble proteins. Nuclear organelles have been actively investigated for their role in cellular regulation and disease. However, these studies are highly challenging to perform in live cells, and therefore, their physico-chemical properties are still poorly understood. In this study, we describe a fluorescence lifetime imaging approach for real-time monitoring of protein condensation in nuclear organelles of live cultured cells. This approach unravels surprisingly large cyclic changes in concentration of proteins in major nuclear organelles including nucleoli, nuclear speckles, Cajal bodies, as well as in the clusters of heterochromatin. Remarkably, protein concentration changes are synchronous for different organelles of the same cells. We propose a molecular mechanism responsible for synchronous accumulations of proteins in the nuclear organelles. This mechanism can serve for general regulation of cellular metabolism and contribute to coordination of gene expression.

Recent developments in Raman spectroscopy instrumentation and data processing algorithms have led to the emergence of Ramanomics - an independent discipline with unprecedented capabilities to map the distribution of distinct molecular groups in live cells. Here, we introduce a method for probing the absolute concentrations of proteins, RNA and lipids in single organelles of live cultured cells by biomolecular component analysis using microRaman data. We found significant cell-to-cell variations in the molecular profiles of organelles, thus providing a physiologically relevant set of markers of cellular heterogeneity. At the same cell the molecular profiles of different organelles can strongly correlate, reflecting tight coordination of their functions. This correlation was significant in WI-38 diploid fibroblasts and weak in HeLa cells, indicating profound differences in the regulation of biochemical processes in these cell lines.

Fluorescence lifetime imaging (FLIM) is an effective noninvasive bioanalytical tool based on measuring fluorescent lifetime of fluorophores. A growing number of FLIM studies utilizes genetically engineered fluorescent proteins targeted to specific subcellular structures to probe local molecular environment, which opens new directions in cell science. This paper highlights the unconventional applications of FLIM for studies of molecular processes in diverse organelles of live cultured cells.