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Nucleic acid synthesis is spatially organized in many organisms. In bacteria, however, the spatial distribution of transcription remains obscure, owing largely to the diffraction limit of conventional light microscopy (200-300 nm). Here, we use photoactivated localization microscopy to localize individual molecules of RNA polymerase (RNAP) in Escherichia(More)
We created a high-throughput modality of photoactivated localization microscopy (PALM) that enables automated 3D PALM imaging of hundreds of synchronized bacteria during all stages of the cell cycle. We used high-throughput PALM to investigate the nanoscale organization of the bacterial cell division protein FtsZ in live Caulobacter crescentus. We observed(More)
The analysis of structure and dynamics of biomolecules is important for understanding their function. Toward this aim, we introduce a method called 'switchable FRET', which combines single-molecule fluorescence resonance energy transfer (FRET) with reversible photoswitching of fluorophores. Typically, single-molecule FRET is measured within a single(More)
Super resolution microscopy such as STORM and (F)PALM is now a well known method for biological studies at the nanometer scale. However, conventional imaging schemes based on sparse activation of photo-switchable fluorescent probes have inherently slow temporal resolution which is a serious limitation when investigating live-cell dynamics. Here, we present(More)
Single-molecule FRET (smFRET) has long been used as a molecular ruler for the study of biology on the nanoscale (∼2-10 nm); smFRET in total-internal reflection fluorescence (TIRF) Förster resonance energy transfer (TIRF-FRET) microscopy allows multiple biomolecules to be simultaneously studied with high temporal and spatial resolution. To operate at the(More)
Histograms of single-molecule Förster resonance energy transfer (FRET) efficiency are often used to study the structures of biomolecules and relate these structures to function. Methods like probability distribution analysis analyze FRET histograms to detect heterogeneities in molecular structure, but they cannot determine whether this heterogeneity arises(More)
During the past decade, localization microscopy (LM) has transformed into an accessible, commercially available technique for life sciences. However, data processing can be challenging to the non-specialist and care is still needed to produce meaningful results. PALMsiever has been developed to provide a user-friendly means of visualizing, filtering and(More)
Although free-living and obligate intracellular bacteria are both polarized it is unclear whether the underlying polarization mechanisms and effector proteins are conserved. Here we dissect at the cytological, functional and structural level a conserved polarization module from the free living α-proteobacterium Caulobacter crescentus and an orthologous(More)