Dr. Markus Grabenbauer
Development of advanced microscopic methods
The correlation of light and electron microscopy is a powerful tool in biomedical research as it combines the investigation of dynamic processes in vivo with the resolution power of the electron microscope. Photo-oxidation is the most direct technique to correlate fluorescent signals to their ultrastructure environment in 3D as detected by electron microscopy and electron tomography.
In our research group, we develop advanced correlative microscopy techniques to understand morpho-functional relationships at molecular level. The green fluorescent proteins (GFPs) and its derivatives revolutionized live-cell light microscopy. Our recently developed protocol with advanced sensitivity, allows for efficient DAB photo-oxidation through GFP and its variants. The labelling resolution is around 5 nm, and the DAB precipitation density is highly correlated to the initial fluorescence intensity at light microscope level, allowing for quantitative studies in electron microscopy and tomography.
The next challenge will be to use the new technologies in neurobiological research to improve understanding of biological structure and function relationships.
Fig.: Correlative microscopy of GFP through photo-oxidation of DAB: From GFP-tagged Golgi enzymes in live cells to their spatial analysis using electron tomography. Adapted from Grabenbauer et al. (2005)
Nat Methods 2:857-862
Movie: Electron tomography of a Golgi stack containing DAB precipitate, which represents the Golgi-resident enzyme GalNAc-T2EGFP. It starts with a ‘virtual flight' through the tomogram in z-axis direction, followed by the manual tracing of membranes to yield a 3D representation of the Golgi stack. The final model of the organelle shows GFP-containing cisternae in green and – highlighted in red – peri-Golgi vesicles found to contain Golgi-resident enzymes. Adapted from Grabenbauer et al. (2005) Nat Methods 2:857-862