Establishing Riboglow-FLIM to visualize noncoding RNAs inside live zebrafish embryos

Area of Science:

• Molecular Biology
• Cell Biology
• Developmental Biology

Background:

• Accurate visualization of RNA in living systems is crucial for understanding cellular functions and disease mechanisms.
• Live zebrafish embryos serve as a valuable model for studying multicellular complexity and disease.
• Current RNA visualization techniques in whole organisms are limited, necessitating advanced imaging tools.

Purpose of the Study:

• To establish and evaluate the Riboglow-FLIM platform for robust RNA visualization in live biological systems.
• To adapt and optimize the Riboglow-FLIM platform for imaging RNA within whole zebrafish embryos.
• To demonstrate the capability of Riboglow-FLIM in resolving cellular heterogeneity of RNA populations in vivo.

Main Methods:

• Systematic evaluation of Fluorescence Lifetime Imaging Microscopy (FLIM) capabilities within the Riboglow platform.
• Utilizing adherent mammalian cells for initial RNA visualization and optimization.
• Injecting labeled mammalian cells into zebrafish embryos for cell-by-cell analysis in a whole-organism context.
• Employing a far-red Cy5-based variant of the Riboglow platform for enhanced imaging in zebrafish.

Main Results:

• Quantitative assessment of Riboglow-FLIM parameters confirmed its efficacy for RNA imaging.
• Successful detection and robust quantification of a model noncoding RNA within live zebrafish embryos.
• Demonstrated ability to resolve cell-to-cell heterogeneity in RNA populations using the developed platform.
• Validation of the platform's applicability in a complex multicellular model.

Conclusions:

• Riboglow-FLIM provides a powerful and quantitative method for visualizing RNA in live zebrafish embryos.
• The platform overcomes limitations in current whole-organism RNA imaging techniques.
• This technology holds significant promise for diverse research fields, including developmental biology and disease modeling.