High-speed, super-resolution live cell imaging system with photostimulation

• Subject Area: Neurosciences
• Term: Funded in 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 545038525

Many dynamic cellular processes, including communication between nerve cells, gene regulation, cellular phase separation, and pathogen invasion, are invisible to conventional fluorescence microscopy because they occur rapidly (hundreds of milliseconds or faster) and over length scales of tens to hundreds of nanometres. Numerous approaches to increase the detail in images of biological cells, resolving objects beyond the Abbe diffraction limit (about 250 nm), have been proposed and are termed Superresolution Microscopy. These methods have found great success in revealing the fine details of cellular structures in fixed samples. However, this success is tempered by the general difficulty to use these methods in living cells, because they are either slow, or need special buffers and/or fluorophores. Another practical difficulty is that resolving dynamic processes beyond the Abbe limit requires collecting time series of superresolved images, which few microscope designs, commercial or otherwise, can achieve before irreversible bleaching washes away signals of interest. Structured illumination microscopy, using Moiré or other patterns to extract information below the diffraction limit, is an attractive approach to obtain super-resolution fluorescence images from live cells. The latest systems use live optical reconstruction, and so avoid slow, error-prone algorithmic assembly from multiple images (and associated bleaching). Rapid (~100 Hz) multicolour time series imaging is thus possible for arbitrary fluorophores across the visible spectrum, with very little bleaching. Some SIM designs are simple enough to mount on commercial microscope stands and can therefore be deployed in parallel with photomanipulation, essential for interrogating biological systems with spatial and temporal precision using optogenetics. With this application we wish to bring this latest superresolution structured illumination technology, combined with photostimulation, to the HU-Berlin. This kind of microscope is not available at all in Berlin. The combination with photomanipulation results in a system with extraordinary capabilities to investigate dynamic cell biology. As outlined in the included research statements, filling this gap in research infrastructure at the HU-Berlin will permit several transformative research projects to answer pressing questions in cellular dynamics, across microbiology, infection, molecular cell biology, gene expression, neurobiology and biophysics, beyond the diffraction limit.

• DFG Programme: Major Research Instrumentation
• Major Instrumentation: Hochgeschwindigkeits Super-Resolution Live-Zell Bildgebungssystem mit Photostimulation
• Instrumentation Group: 5090 Spezialmikroskope
• Applicant Institution: Humboldt-Universität zu Berlin
• Leader: Professor Dr. Andrew Plested