Single-molecule super-resolution


Super-resolution microscopy is a noninvasive technology that enables living cells to be visualized with resolution finer than the wavelength of light. Since its advent, super-resolution has revolutionized biological imaging and the life sciences. There are two major "flavors" of super-resolution imaging. One variety (Stimulated emission depletion, or STED microscopy) involves structuring how a sample is illuminated, and targeted readout of fluorescence emission from sub-diffraction limited imaging volumes. On the other hand, my own research achieves super-resolution by detecting single fluorescent molecules within a sample of interest, and then spatially localizing detected molecules with ~25 nanometer precision. The figure below illustrates some of the key ideas.


Super-resolution in a nutshell. (a) Schematic of a single-molecule super-resolution imaging system. By reducing the concentration of actively emitting fluorescent molecules, individual molecules are visible on a camera sensor. (b) Even though the fluorescent molecules are smaller than a couple nanometers in size, the images that they form on the camera are "diffraction-limited" and appear to be a few hundred nanometers in width. By fitting pixilated camera images to Gaussian model functions, the true underlying positions of the molecules are estimated. (c) When a large ensemble of fluorescent molecules densely label a biological structure, relevant detail is obscured. However, by limiting the concentration of actively fluorescing molecules, and localizing molecules sequentially, a super-resolution image is reconstructed by plotting the coordinates of the localized molecules.

Below, we illustrate a real-world example of super-resolution imaging. A conventional microscope image (scale bar is 2 microns) is shown of the microtubules of a single cell, labeled with a fluorescent dye. The image appears quite blurry due to optical diffraction, a phenomena caused by the wave-nature of light.


A diffraction-limited microscope image of a cell's microtubules.

By treating the cell with a finely-tuned cocktail of chemicals, and imaging it at high laser intensities, individual fluorescent molecules are observed "blinking" on and off. These molecules are subsequently localized, and their positions are plotted.


The video above illustrates the super-resolution reconstruction process. The panel on the left shows the raw single-molecule data that is collected over the course of a super-resolution experiment, while the data on the right plots the coordinates of localized single molecules. As more molecules are detected, a finely-sampled super-resolution image gradually appears. (Data and super-resolution reconstruction video courtesy of Dr. Matthew Lew.)