Characterization of LLPS Using Super-Resolution Imaging

Microscopy is an effective tool for visualizing the structure and composition of biomolecular condensates. Based on the cutting-edge super-resolution fluorescence microscopy platform, CD BioSciences offers customized experimental procedures to help you obtain super-resolution images of liquid-liquid phase separation (LLPS) condensates under different conditions.

LLPS, an emerging phenomenon, is a potential mechanism for assembling biomolecular condensates. There are two types of multivalent interactions that lead to LLPS. One is the regular multivalent interactions between proteins and RNA with each other. The other is a weak, transient, multivalent interaction between intrinsically disordered regions (IDRs). Depending on the strength of the interaction, the droplet size varies. Typically conventional wide-field fluorescence imaging can easily distinguish and characterize huge droplets. However, for small droplets beyond the diffraction limit, conventional imaging techniques cannot achieve accurate quantification. The advent of super-resolution microscopy has enabled scientists to understand cell biology in greater detail and can reveal functional interrogations of physicochemical parameters within cells at the nanoscale.

Customized Services

Our super-resolution microscopy platform is widely applicable to LLPS analysis of biomolecular condensates of small size (1-2 μm). CD BioSciences has a whole range of super-resolution microscopy methods, including:

• Stimulated Emission Dissipation Microscopy (STED)
• Structural Illumination Microscopy (SIM)
• Photoactivated Localization Microscopy (PALM)
• Stochastic Optical Reconstruction Microscopy (STORM)

These methods can achieve resolutions of 10-20 nm in the horizontal plane and 10-75 nm in the cross-sectional plane, and have been applied to the analysis of many biological studies, including phase-separated condensates. In addition, we provide a unique super-high resolution microscopy system to enhance the resolution of characterized LLPS. This approach is associated with specific dyes and photoactivated proteins to label target structures, long time and labor-intensive sample preparation. Here, CD BioSciences provides a detailed protocol for labeling, imaging and analyzing LLPS condensates using super-resolution microscopy.

(1) Sample preparation.

(2) Microscope setup.

(3) Image acquisition.

(4) Image analysis and quantification.

(5) LLPS condensate identification and segmentation.

Advantages of the Super-Resolution Imaging Platform

• Allows the localization of each molecule in small condensates smaller than the optical diffraction limit.
• Provides information on the dynamics of molecules in LLPS condensates, allowing characterization of the diffusion behavior of molecules in the condensed and dilute phases.
• For characterizing the biochemical function and assembly mechanism of cohesions within diffraction-limited foci in bacteria.
• For observing the formation of LLPS condensates during transcription.

Following the above protocol, we not only help you to obtain super-resolution images of LLPS condensates under different conditions, but also further analyze the statistical information of condensed molecules from different groups, including the localization of each condensate, the number of condensates per cell, and the condensation region. If you are interested in our services, please do not hesitate to contact us for more information.