When Assistant Professor of Chemistry Ke Xu finished his Ph.D. on superconductors, he decided to work in a different research field that is also “super:” super-resolution microscopy, a name collectively given to a variety of rising new microscopy techniques that achieve nanometer-scale spatial resolutions with light.
Xu, a Chan-Zuckerberg Biohub investigator, is pushing the frontiers of super-resolution microscopy to enable the probing of functional parameters — including pH, chemical polarity, and molecule diffusivity — at the nanometer scale. To achieve this, his lab is adding novel dimensions to super-resolution microscopy beyond the traditional 3D spatial dimensions. (Image 1)
In one case, he has successfully added in the spectral (color) dimension by integrating single-molecule spectroscopy. As a result, he has created a method to measure the fluorescence spectra of millions of individual molecules in just a few minutes, achieving spectrally resolved super-resolution microscopy, a first for multicolor imaging. (Image 2)
Then, by applying this technique to fluorescent molecules capable of reporting local environments through spectral (color) changes, he has unveiled amazing compositional and property heterogeneities at the nanometer scale, both for live mammalian cells (Image 3) and for surface chemistry (Image 4).
Super-resolution image with regular 3D spatial dimensions of neural stem cell (Hauser et al. 2018)
- The colors represent the height information in this 3D super-resolution image, with blue/violet being close to the underlying glass substrate and green/yellow being far away.
- Color variations indicate variations in the height of the membrane cytoskeleton from the substrate.
- This image labels spectrin, a component of the membrane cytoskeleton of the cell.
Spectrally resolved, multicolor super-resolution image of a mammalian fibroblast cell (Zhang et al. 2015)
- Four far-red dyes, with slight differences in emission spectrum, were used to label the four different intracellular structures.
- The colors represent the measured spectral peak positions of individual molecules on a continuous scale (676-720 nm in wavelength).
- The four major colors showed up due to the four dyes used.
- The colors highlight the following — magenta: mitochondria, cyan: microtubules, green: vimentin filaments, and yellow: peroxisomes.
Spectrally resolved, polarity-sensing super-resolution image of the membranes in a mammalian fibroblast cell (Moon et al. 2017)
- Nile Red, a solvatochromic dye that changes color (spectrum) based on local chemical polarity, is used in this spectrally resolved super-resolution study, which labeled all the membranes of the cell.
- Each detected single molecule is color-coded according to its spectrum peak position, on a continuous scale of 612-648 nm.
- Spectral resolution shows polarity differences between organelle and plasma membranes in the living cell; such differences are due to a disparity in cholesterol levels.
Spectrally resolved, polarity-sensing super-resolution image of microscopic organic droplets on a glass surface (Xiang et al. 2018)
- For this image trichloroethylene (TCE) and chloroform spontaneously demixed on a glass surface to form adlayer nanodroplets of different compositions.
- Nile Red, a solvatochromic dye that changes color (spectrum) based on local chemical polarity, is used in this spectrally resolved super-resolution study.
- Each detected single molecule is color-coded according to its spectrum peak position, on a continuous scale of 620-638 nm.
- A palette of different colors and nanoscale structures was found for the adsorbed organic droplets, thus revealing unexpectedly rich structural and compositional behaviors of surface adlayers at the nanoscale.
Hauser, M., Yan, R., Li, W., Repina, N. A., Schaffer, D. V. and Xu, K., 2018. “The spectrin-actin-based periodic cytoskeleton as a conserved nanoscale scaffold and ruler of the neural stem cell lineage.” Cell Rep., 24: 1512-1522.
Moon, S., Yan, R., Kenny, S. J., Shyu, Y., Xiang, L., Li, W. and Xu, K., 2017. “Spectrally resolved, functional super-resolution microscopy reveals nanoscale compositional heterogeneity in live-cell membranes.” J. Am. Chem. Soc., 139: 10944-10947.
Xiang, L., Wojcik, M., Kenny, S. J., Yan, R., Moon, S., Li, W. and Xu, K., 2018. “Optical characterization of surface adlayers and their compositional demixing at the nanoscale.” Nat. Commun., 9: 1435.
Zhang, Z., Kenny, S. J., Hauser, M., Li, W. and Xu, K., 2015. “Ultrahigh-throughput single-molecule spectroscopy and spectrally resolved super-resolution microscopy.” Nature Methods, 12: 935-938.