Biomolecular condensates — membraneless, microscopic structures that concentrate proteins and other molecules in cells — are crucial to the organization of cellular biochemistry. Insight into the development and behavior of condensates could lead to better treatments for infectious diseases, cancer, and neurological disorders. Researchers at New York University (NYU) aimed to measure condensate composition and dynamics without relying on conventional techniques, like fluorescence labeling or surface attachment, which can damage fragile condensate samples. Until now, scientists have needed to distort condensate samples to study them. “It’s been the elephant in the room for scientists,” professor Saumya Saurabh said. “Our research provides a precise and noninvasive way to study biomolecular condensates.” To overcome the limitations of conventional techniques, the team used label-free holographic microscopy to investigate the behavior of a condensate-forming protein in vitro. The researchers flowed thousands of droplets through a holographic microscope in a microfluidic channel to visualize and characterize each particle individually. The holographic characterization was free from perturbations and was able to gather data on thousands of particles in minutes. Precise information about the droplet’s size, shape, and refractive index was encoded in the hologram of each μm-scale droplet. This image offers a look inside biomolecular condensates, the tiny, membraneless compartments within cells. The circular visualization represents a condensate, with left half (colorful) showing intricate nanoscale organization as revealed by superresolution microscopy. The right half (concentric rings) represents holographic microscopy, which provides precise measurements of the condensate’s overall composition and dynamic behavior. Together, these advanced imaging methods, along with molecular simulations (top right structure) and mathematical models (bottom left equation), are helping researchers unlock the complex secrets of these essential cellular structures. Courtesy of Julian von Hofe and Saumya Saurabh. The researchers used this technique to examine PopZ, a condensate-forming protein that influences cell growth. The precision and speed provided by the digital holography technique enabled the team to monitor the kinetics of the condensate’s formation, growth, and aging over time. By systematically varying the concentration and valence of cations, the researchers found that multivalent ions influence condensate organization and dynamics. “I was surprised by their complex and incredibly sensitive response to different ionic species,” researcher Julian von Hofe said. “Even a small change in ionic valency drastically altered both condensate concentration and dynamics.” The researchers used superresolution microscopy to explore the architecture of PopZ at the nanoscale. Data acquired through superresolution imaging revealed that the condensates were not uniform droplets, but exhibited intricate nanoscale organization, and that PopZ droplet growth deviated from classical models. These findings were supported by molecular dynamics simulations, which provided atomic-level insights into the biocondensate assemblies. The study thus demonstrated the value of holographic microscopy as a hypothesis-generating tool that provides noninvasive insight into condensate substructure, that can be further tested and refined using complementary, minimally perturbative methods. “Being able to see ‘under the hood’ for the first time has revealed some big surprises about this important class of systems,” professor David Grier said. Although the researchers observed the condensates in vitro, their findings could contribute to a more complete understanding of condensate behavior within living cells. “The intricate reality of biomolecular condensates, as revealed by our findings, goes far beyond simple liquid-liquid phase separation,” Saurabh said. A better understanding of how biomolecular condensates are organized and grow, made possible through holographic microscopy and superresolution imaging, could help shape disease modeling and future drug development. For example, the proteins that form plaques in ALS are fluid condensates in good health. “Understanding how a spherical condensate forms into a deadly plaque is an opportunity to better understand ALS,” Saurabh said. The biomolecular condensates in the cells can also house drug molecules that are intended for a different purpose. This phenomenon could help explain why drugs that are designed to target a specific protein still cause side effects. By using holographic microscopy to analyze condensate dynamics with extreme precision, scientists can identify the subtle differences in condensate composition and architecture that occur when drug molecules enter a condensate. “For example, we can now explore the chemical space of drug modifications to precisely control their partitioning, achieving the specificity needed to prevent them from entering condensates,” Saurabh said. “This opens new avenues for how we think about designing drugs and their potential side effects.” This work highlights the power of holographic microscopy, especially when used with superresolution imaging, to probe the properties and mechanistic underpinnings of biomolecular condensates. “Our collaboration has introduced fast, precise, and effective methods for measuring the composition and dynamics of macromolecular condensates,” Grier said. The research was published in the Journal of the American Chemical Society (www.doi.org/10.1021/jacs.5c02947).