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Additional file 1: Figure S1. Single expression vector maps and sequences of the designed linkers. Figure S2. Unsuccessful attempt for MT labeling using Met-TAG-α-tubulin. Figure S3. Unsuccessful attempt of labeling of MVBs, Exosomes and ER using the GCE-tag and SiR-Tet labeling. Figure S4. Unsuccessful mitochondria labeling using the GCE-tag. Figure S5. Labeling cellular proteins using the GCE-tag. Figure S6. Evaluating peroxisomes, exosomes and MTs labeling using Flag/Myc- GGSG-TAG or GGSG-TAG as GCE-tags. Table S1. Sequences of organelle markers used in this work.

Tracking the localization and mobility of individual proteins in live cells is key for understanding how they mediate their function. Such information can be obtained from single molecule imaging techniques including as Single Particle Tracking (SPT) and Single Molecule Localization Microscopy (SMLM). Genetic code expansion (GCE) combined with bioorthogonal chemistry offers an elegant approach for direct labeling of proteins with fluorescent dyes, holding great potential for improving protein labeling in single molecule applications. Here we calibrated conditions for performing SPT and live-SMLM of bioorthogonally labeled plasma membrane proteins in live mammalian cells. Using SPT, the diffusion of bioorthogonally labeled EGF receptor and the prototypical Shaker voltage-activated potassium channel (Kv) was measured and characterized. Applying live-SMLM to bioorthogonally labeled Shaker Kv channels enabled visualizing the plasma membrane distribution of the channel over time with ∼30 nm accuracy. Finally, by competitive labeling with two Fl-dyes, SPT and live-SMLM were performed in a single cell and both the density and dynamics of the EGF receptor were measured at single molecule resolution in subregions of the cell. We conclude that GCE and bioorthogonal chemistry is a highly suitable, flexible approach for protein labeling in quantitative single molecule applications that outperforms current protein live-cell labeling approaches.

The progressive nature of the dementia of Alzheimer's disease has significant clinical, functional, legal and nursing implications. Traditionally, the grading of severity of the disease is based on evaluation of the patients' independence and competence. However, the term competence has been used in different respects, leading to obscuration and confusion. Moreover, the evaluation of competence is based on different tests and there is no single accepted method for this purpose. In the absence of such a criterion, there is a need for an objective test to evaluate the functional competence of the patient. This will be based on complex behavioral activities and instrumental activities of daily living in real situations, in the natural environment of the patient. The limits of available methods are discussed with practical suggestions for the development of such a test.