Spectroscopy/Super-resolution Microscopy: Fine spectral resolution and single-molecule imaging - fast

A combination of spectroscopy and microscopy is enabling fast imaging of single molecules with unprecedented spectral and spatial resolution.1 Spectrally resolved stochastic optical reconstruction microscopy (SR-STORM) allows high-resolution imaging of multiple components and local chemical environments (such as pH variations) within a cell. The high-throughput technique can provide full spectral and spatial information on millions of single molecules in about five minutes.

(L-R) Samuel Kenny, Zhengyang Zhang, Ke Xu, Margaret Hauser, and Wan Li have invented a new technology to image single molecules with unprecedented spectral and spatial resolution
(L-R) Samuel Kenny, Zhengyang Zhang, Ke Xu, Margaret Hauser, and Wan Li have invented a new technology to image single molecules with unprecedented spectral and spatial resolution. (Credit: Roy Kaltschmidt/Berkeley Lab)

The achievement builds on the work that Ke Xu, a faculty scientist in the Life Sciences Division at Lawrence Berkeley National Laboratory (California), did as a postdoctoral researcher at Harvard University (Cambridge, MA) with Xiaowei Zhuang, who invented STORM—a super-resolution method based on single-molecule imaging and photoswitching.

By devising a dual-objective system with two microscope lenses facing each other, Xu and colleagues viewed the front and back of a specimen at the same time to achieve optical resolution of approximately 10 nm. The dual-objective system disperses "the single-molecule image collected by one objective lens into spectrum while keeping the other image for single-molecule localization," Xu says.

The innovation promises to facilitate understanding of the behavior of individual molecules, and enable high-quality multicolor imaging of multiple targets. Using this method to image neurons, the team showed that actin, a key component of the cytoskeleton, has a different structure in axons than in dendrites.

Testing the technique, the researchers dyed a sample with 14 dyes in a narrow emission window and excited and photoswitched the molecules with one laser. While the dyes' spectra are heavily overlapping, the researchers report being surprised by how easy the molecules were to identify.

While applications are initially in fundamental research and cell biology, Xu hopes to impact medical applications, noting that the technique may show whether degradation of cell structures correlate with disease. Xu is now working to refine the method, make it work with conventional microscope systems (for broader accessibility), and to develop new dyes and probes for monitoring the environment within living cells.

1. Z. Zhang, S. J. Kenny, M. Hauser, W. Li, and K. Xu, Nat. Methods (2015); doi:10.1038/nmeth.3528.

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