Polarization-Encoded Colocalization Microscopy at Cryogenic Temperatures

Abstract

Super-resolution localization microscopy is based on determining the positions of individual fluorescent markers in a sample. The major challenge in reaching an ever higher localization precision lies in the limited number of collected photons from single emitters. To tackle this issue, it has been shown that one can exploit the increased photostability at low temperatures, reaching localization precisions in the subnanometer range. Another crucial ingredient of single-molecule super-resolution imaging is the ability to activate an individual emitter within a diffraction-limited spot.Here, we report on the photoblinking behavior of organic dyes at low temperature and elaborate on the limitations of this ubiquitous phenomenon for selecting single molecules. We then show that recording the emission polarization not only provides access to the molecular orientation, but it also facilitates the assignment of photons to individual blinking molecules. Furthermore, we employ periodical modulation of the excitation polarization as a robust method to effectively switch fluorophores. We benchmark each approach by resolving two emitters on different DNA origami structures.

Deciphering a hexameric protein complex with Angstrom optical resolution

“…The histogram in Fig. 2d plots the distribution of the side lengths of the projected triangles and is well-described by a fit that considers the localization uncertainty as well as the random particle orientation (14). We determine a side length of 9.9 ± 0.6 nm in excellent agreement with the expected value.…”

“…These studies have shown that PCNA forms a stable homo-trimer with a pseudo-hexameric shape. The stability and simple configuration of its structure makes PCNA a good model system for benchmarking our imaging methodology (14). To study Human PCNA with polarCOLD, we first fully labeled it via a His-tag linker on the N-terminal side of each subunit of the protein, forming an equilateral triangle (see method section and Fig.…”

“…S1). PCNA complexes were then embedded in a hydrophilic poly-vinyl alcohol (PVA) matrix at sub-nanomolar concentration and spin-coated on a mirror-enhanced substrate (14). The resulting density corresponds to fewer than one protein per m 2 on average so that individual proteins can be easily resolved in diffraction-limited imaging.…”

“…Then 2 µl of the protein was mixed with the 360 µl PVA solution and filtered with a 100 nm spin filter (Whatman Anotop-10). 5 µl of this mixed solution was then spin coated (30 s at 1000 rpm followed by 3000 rpm for 60 s) onto a plasma-cleaned mirror-enhanced substrate, prepared in house, (14), and immediately loaded into our custom-built cryogenic microscope.…”

“…In one implementation, cryogenic optical localization in 3D (COLD) was introduced, where the stochastic intensity blinking of organic dyes gave access to the positions of up to four labeling sites on a single protein (21). Recently, we employed a more robust protocol to identify individual fluorophores by exploiting the polarization of the fluorescence light dictated by the fluorophore orientation (14). This latter method was validated by measuring single distances on one-dimensional DNA nanorulers.…”