Characterization of LLPS Using Confocal Microscopy

Microscopy is an effective tool for visualizing the structure and composition of biomolecular condensates. Based on the cutting-edge confocal microscopy platform, CD BioSciences offers customized experimental procedures to quantify liquid-liquid phase separation (LLPS) in cell-generated biomolecular condensates with minimal temporal resolution within milliseconds.

Biomolecules may undergo liquid-liquid phase separation (LLPS) to compartmentalize and regulate different biological processes spatiotemporally. Visualization limited to membraneless organelles is not sufficient when studying the phase separation of biomolecular condensates in cellulose. Due to the dynamic process of assembly/disassembly of such structures, a large amount of data with high spatiotemporal resolution needs to be accumulated when analyzing living cells. Many experimental techniques are currently used to study the process of LLPS, but fluorescence microscopy of biomolecular condensates containing fluorescently labeled components has emerged as a dominant method. Among them, confocal fluorescence microscopy provides great possibilities for studying LLPS in biomolecular condensates.

Customized Services

Our confocal microscopy platform is widely applicable to the analysis of LLPS in cell-generated biomolecular condensates in the 200 nm-1 mm range. With confocal fluorescence imaging, CD BioSciences provides you with more detailed information about biomolecular condensates, including:

✓ Obtain real-time 2D and 3D images of biomolecular condensates.

✓ Determine the number, location and fluorescence intensity of biomolecular condensate scaffold proteins visible in concentrated and diluted states.

✓ We use the calibration dependence of the fluorescence intensity of visible proteins on their concentration to determine the amount of scaffold proteins in the concentrated and diluted phases of biomolecular condensates.

To attract customers, our team of experts is developing the use of confocal fluorescence microscopy in the spinning disk confocal fluorescence microscopy variant to increase the amount of data acquired and enable information to be collected from 1000 parallel spots per unit of time. In addition, we offer the use of confocal fluorescence microscopy in selective planar illumination microscopy and lattice light sheet microscopy to reduce photodamage to the sample. CD BioSciences offers the following confocal fluorescence strategies to help customers analyze biomolecular condensate interactions.

• Fluorescence Recovery After Photobleaching (FRAP) Strategy
  We offer the use of confocal fluorescence microscopy in variant FRAP to determine the droplet properties of biomolecular condensates in cellulose. In addition, our FRAP analysis allows the determination of the ratio of mobile to immobile molecules in the analyzed condensates.
• Fluorescence Correlation Spectroscopy (FCS) Strategy
  Our FCS method is widely used in LLPS studies, including the determination of the concentration and diffusion coefficients of individual molecules in biomolecular condensates. This method primarily uses confocal microscopy to record the temporal traces of fluorescence from refractionally restricted sites, and is applicable both in vivo and ex vivo.

Advantages of the Confocal Microscopy Platform

• High contrast.
• High resolution and sensitivity.
• Short acquisition time.
• Suitable for analyzing the localization of fluorescently labeled macromolecules within membraneless organelles and monitoring morphological changes within them.
• Widely used to study phase-separated droplets prepared in vitro.

CD BioSciences offers comprehensive confocal fluorescence strategies for studying RNA-protein condensates. Our team of experts has developed detailed protocols for preparing confocal slides, performing the experiments described above, and interpreting the resulting data. If you are interested in our services, please do not hesitate to contact us for more information.