Cellular processes are orchestrated by many biomolecules operating in a spatially and temporally coordinated manner within a tiny volume. To uncover the organizational principles underlying these processes and their functional relevance using microscopy visualization, we have developed a fragment-tagging approach that fluorescently labels target proteins using our engineered split-fluorescent proteins. This approach has enabled systematic generation of knockin cell lines with endogenously labeled proteins through CRISPR/Cas9-mediated gene editing. We have also developed the epi-illumination selective plane illumination microscopy (eSPIM) light-sheet microscopy platform, enabling high-resolution, high-throughput imaging of multiple cell lines. Combining these two techniques has paved the way for systematic mapping of the spatial localization and temporal dynamics of proteins in living cells. In the closing section of the webinar, we will focus on electron-multiplying charge-coupled device (EMCCD) imaging technology involved in proteome research. Protein-driven phenomena tend to happen at very small scales of space and time. These challenging environments call for advanced light manipulation and detection technologies optimized for low-light conditions. We will discuss the outlook for future camera-based imaging for applications aiming to uncover gene position, activity, and the resultant protein’s role and fate.
During this webinar, the speakers will:
- Explain split-fluorescent protein tags for efficient labeling of endogenous proteins in cell lines
- Describe the eSPIM system for high-throughput volumetric imaging of living cells
- Discuss the perspective of library-based fluorescence microscopy analysis as a primary discovery tool
- Highlight selected technical aspects of imaging-camera performance relevant for efficient visualization of gene expression as part of the proteome analysis of living cells
- Answer viewer questions live during the broadcast.
This webinar will last for approximately 60 minutes.