Characterization of LLPS Using Electron Microscopy

Microscopy is an effective tool for visualizing the structure and composition of biomolecular condensates. Based on the cutting-edge electron microscopy platform, CD BioSciences offers customized experimental procedures to precisely observe and dynamically image liquid-liquid phase separation (LLPS) at nanoscale spatial resolution in a label-free manner.

LLPS or coalescence is a ubiquitous phenomenon involving many intracellular and extracellular biological processes. While it is well established that LLPS can be described as the breakdown of spinodal lines leading to the delamination of initially homogeneous protein solutions, little is known about the assembly pathways by which soluble proteins aggregate into dense microdroplets. Electron microscopy employs a beam of accelerated electrons to illuminate a fixed contrast-stained sample and record images at a spatial resolution as low as 0.8 Å in a highly ordered structure. Scientists have successfully employed transmission electron microscopy to observe the phase separation of intrinsically disordered proteins occurring in a liquid environment.

Customized Services

Our transmission electron microscopy (TEM) platform is widely used to analyze membrane-free organelles in cells such as nucleoli, nuclear speckles, Cajal bodies and RNP particles, as well as separated components of macromolecular phases formed in vitro, such as hydrogels and protofibrils. With this approach, we can easily help you capture the nucleation and initial growth steps of LLPS.

To obtain information on the localization and spatial distribution of biomolecular condensates using TEM, we typically immunocouple the sample with colloidal gold particles coated with specific antibodies. Here, CD BioSciences provides comprehensive electron microscopy techniques to better understand the steps involved in LLPS at the nanoscale in a time-resolved manner.

• Electron Spectroscopic Imaging (ESI)
  Negative staining in conventional TEM allows for high contrast, but cellular components may respond differently to the stain, potentially leading to non-uniform labeling of the specimen. Here, we offer an improved TEM, called electron spectroscopic imaging (ESI), to distinguish nucleic acids and proteins in membrane-free organelles by exploiting differential energy loss upon irradiation of naturally abundant elements.
• Cryogenic Transmission Electron Microscopy (cryoTEM)
  We offer cryoTEM to image coalescing microdroplets at submicron resolution, which can provide you with information on the final structure produced by LLPS.
• Direct Electron Detector for Recording TEM Images
  We offer direct electron detectors for recording TEM images with higher signal-to-noise ratios and fast frame rates to enable in situ or direct observation of LLPS condensate nucleation and growth, and to track processes such as gold nanoparticle growth, polymerization-induced colloid formation, and lysozyme aggregation.
• Light and electron microscopy (CLEM)
  We offer CLEM as a complementary method to gain insight into the structural and dynamic properties of agglomerates. Our expert team has successfully developed CLEM to image the ultrastructure of nucleolus subcompartments and to track specific proteins found in cytoplasmic inclusions.

Advantages of the Electron Microscopy Platform

• Requires fixation and processing of samples in a label-free manner prior to imaging.
• Allows direct in situ observation in liquid TEM.
• Allows analysis of the dynamic assembly of intrinsically disordered proteins during LLPS.

Our electron microscopy is a well-established technique that provides clients with structural information on biomolecular condensates, covering medium to sub-nanometer scale spatial resolution. If you are interested in our services, please do not hesitate to contact us for more information.