Anomalous diffusion in crowded fluids, e.g., in the cytoplasm of living cells, is a frequent phenomenon. So far, however, the associated stochastic process, i.e., the propagator of the random walk, has not been uncovered. Here we show by means of fluorescence correlation spectroscopy and simulations that the properties of crowding-induced subdiffusion are consistent with the predictions for fractional Brownian motion or obstructed (percolationlike) diffusion, both of which have stationary increments. In contrast, our experimental results cannot be explained by a continuous time random walk with its distinct non-Gaussian propagator.

We present a scaling formula for size-dependent viscosity coefficients for proteins, polymers, and fluorescent dyes diffusing in complex liquids. The formula was used to analyze the mobilities of probes of different sizes in HeLa and Swiss 3T3 mammalian cells. This analysis unveils in the cytoplasm two length scales: (i) the correlation length ξ (approximately 5 nm in HeLa and 7 nm in Swiss 3T3 cells) and (ii) the limiting length scale that marks the crossover between nano- and macroscale viscosity (approximately 86 nm in HeLa and 30 nm in Swiss 3T3 cells). During motion, probes smaller than ξ experienced matrix viscosity: η(matrix) ≈ 2.0 mPa·s for HeLa and 0.88 mPa·s for Swiss 3T3 cells. Probes much larger than the limiting length scale experienced macroscopic viscosity, η(macro) ≈ 4.4 × 10(-2) and 2.4 × 10(-2) Pa·s for HeLa and Swiss 3T3 cells, respectively. Our results are persistent for the lengths scales from 0.14 nm to a few hundred nanometers.

The growing demands of advanced fluorescence and super-resolution microscopy benefit from the development of small and highly photostable fluorescent probes. Techniques developed to expand the genetic code permit the residue-specific encoding of unnatural amino acids (UAAs) armed with novel clickable chemical handles into proteins in living cells. Here we present the design of new UAAs bearing strained alkene side chains that have improved biocompatibility and stability for the attachment of tetrazine-functionalized organic dyes by the inverse-electron-demand Diels-Alder cycloaddition (SPIEDAC). Furthermore, we fine-tuned the SPIEDAC click reaction to obtain an orthogonal variant for rapid protein labeling which we termed selectivity enhanced (se) SPIEDAC. seSPIEDAC and SPIEDAC were combined for the rapid labeling of live mammalian cells with two different fluorescent probes. We demonstrate the strength of our method by visualizing insulin receptors (IRs) and virus-like particles (VLPs) with dual-color super-resolution microscopy.

Under tension: A set of genetically encoded unnatural amino acids can be used for biocompatible site‐specific labeling of proteins with fluorogenic dyes. The new compounds have norbornene and trans‐cyclooctene units that react with tetrazine derivatives in an inverse‐electron‐demand Diels–Alder cycloaddition (left in picture). The technique offers fast labeling that is orthogonal to labeling through azide–cyclooctyne click reaction (right).