Using light microscopy to visualize cellular structures and dynamics has been an important approach to cell biology research for centuries. However, many subcellular features, for example the morphology of organelles or the 3D organization of chromatin, cannot be resolved by conventional light microscopy. Improving the resolution of light microscopy is therefore a critical need in biological research.
Today, several methods surpass the diffraction limit of light microscopy and achieve down to single-nanometer resolution. In our lab we evolve these methods, pushing the temporal and spatial resolution of fluorescence microscopy far beyond the limits of conventional light microscopy.
DNA-PAINT and FLASH-PAINT
DNA-based fluorescent probes are a powerful tool for super-resolution microscopy and other microscopy modalities. Our lab actively develops new probes and labeling approaches in this field. Our fluorogenic DNA-PAINT probes (Chung et al.,Nature Methods 2022) substantially reduce the background over traditional DNA-PAINT probes and thereby allow for 26-fold faster super-resolution imaging and 3D imaging without the need for optical sectioning. With FLASH-PAINT (Schueder et al., Cell 2024), which allows for the rapid, highly efficient and gentle exchange of fluorescent probes between different targets, we have developed a new method that allows for highly multiplexed DNA-PAINT super-resolution microscopy. We are continuing to push the technology development at this frontier and are actively applying them to a range of biomedical questions.
Instrumentation
We have a long history of developing super-resolution STED and SMLM microscopes. Recent efforts have focused on improving their 3D and multicolor imaging capabilities (Huang*, Sirinakis*, et al., Cell 2016; Zhang*, Schroeder*, et al., Nature Methods 2020; Hao et al. Nature Methods 2021) and increasing imaging depth (Velasco et al., Optica 2021), throughput and degree of automation (Barentine et al., Nature Biotech 2023). We are now correlating super-resolution microscopy with other imaging modalities such as quantitative phase contrast, building a 4Pi-Lattice Light Sheet instrument for better 3D live-cell super-resolution microscopy, developing a light-sheet microscope for pan-ExM samples, and integrating microfluidics into automated microscopes for highly multiplexed FLASH-PAINT imaging.
pan-Expansion Microscopy (pan-ExM)
pan-Expansion Microscopy (pan-ExM) is a super-resolution technique that offers optical contrast equivalent to EM heavy-metal stains. By physically expanding a biological sample about 16 to 24-fold in every dimension, bulk (pan-) staining of proteins or other molecules can reveal general subcellular organization. Using just a confocal microscope, structures down to ~10-40 nm size are resolved. By further combining these pan-stains with antibody labels of specific proteins in multicolor images, images resembling those of correlative light and electron microscopy (CLEM) can be obtained (M’Saad et al., Nature Communications 2020; M’Saad et al., bioRxiv 2022; M’Saad et al., bioRxiv 2022). Our lab is developing this technology for the study of cultured mammalian cells, mouse tissue, human tissue and other biological samples.