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 coli with a spatial resolution of ∼40 nm. In cells growing rapidly in nutrient-rich media, we find that RNAP is organized in 2-8 bands. The band number scaled directly with cell size (and so with the chromosome number), and bands often contained clusters of >70 tightly packed RNAPs (possibly engaged on one long ribosomal RNA operon of 6000 bp) and clusters of such clusters (perhaps reflecting a structure like the eukaryotic nucleolus where many different ribosomal RNA operons are transcribed). In nutrient-poor media, RNAPs were located in only 1-2 bands; within these bands, a disproportionate number of RNAPs were found in clusters containing ∼20-50 RNAPs. Apart from their importance for bacterial transcription, our studies pave the way for molecular-level analysis of several cellular processes at the nanometer scale.

Colocalization of differently labeled biomolecules is a valuable tool in fluorescence microscopy and can provide information on biomolecular interactions. With the advent of super-resolution microscopy, colocalization analysis is getting closer to molecular resolution, bridging the gap to other technologies such as fluorescence resonance energy transfer. Among these novel microscopic techniques, single-molecule localization-based super-resolution methods offer the advantage of providing single-molecule coordinates that, rather than intensity information, can be used for colocalization analysis. This requires adapting the existing mathematical algorithms for localization microscopy data. Here, we introduce an algorithm for coordinate-based colocalization analysis which is suited for single-molecule super-resolution data. In addition, we present an experimental configuration for simultaneous dual-color imaging together with a robust approach to correct for optical aberrations with an accuracy of a few nanometers. We demonstrate the potential of our approach for cellular structures and for two proteins binding actin filaments.

Re-establishment of nuclear structure and chromatin organization after cell division is integral for genome regulation or development and is frequently altered during cancer progression. The mechanisms underlying chromatin expansion in daughter cells remain largely unclear. Here, we describe the transient formation of nuclear actin filaments (F-actin) during mitotic exit. These nuclear F-actin structures assemble in daughter cell nuclei and undergo dynamic reorganization to promote nuclear protrusions and volume expansion throughout early G1 of the cell cycle. Specific inhibition of this nuclear F-actin assembly impaired nuclear expansion and chromatin decondensation after mitosis and during early mouse embryonic development. Biochemical screening for mitotic nuclear F-actin interactors identified the actin-disassembling factor cofilin-1. Optogenetic regulation of cofilin-1 revealed its critical role for controlling timing, turnover and dynamics of F-actin assembly inside daughter cell nuclei. Our findings identify a cell-cycle-specific and spatiotemporally controlled form of nuclear F-actin that reorganizes the mammalian nucleus after mitosis.

The localization precision is a crucial and important parameter for single-molecule localization microscopy (SMLM) and directly influences the achievable spatial resolution. It primarily depends on experimental imaging conditions and the registration potency of the algorithm used. We propose a new and simple routine to estimate the average experimental localization precision in SMLM, based on the nearest neighbor analysis. By exploring different experimental and simulated targets, we show that this approach can be generally used for any 2D or 3D SMLM data and that reliable values for the localization precision σ SMLM are obtained. Knowing σ SMLM is a prerequisite for consistent visualization or any quantitative structural analysis, e.g., cluster analysis or colocalization studies.

Dense fluorophore labeling without compromising the biological target is crucial for genuine super-resolution microscopy. Here we introduce a broadly applicable labeling strategy for fixed and living cells utilizing a short peptide tag-specific nanobody (BC2-tag/bivBC2-Nb). BC2-tagging of ectopically introduced or endogenous proteins does not interfere with the examined structures and bivBC2-Nb staining results in a close-grained fluorophore labeling with minimal linkage errors. This allowed us to perform high-quality dSTORM imaging of various targets in mammalian and yeast cells. We expect that this versatile strategy will render many more demanding cellular targets amenable to dSTORM imaging.

The inheritance of the histone H3 variant CENP-A in nucleosomes at centromeres following DNA replication is mediated by an epigenetic mechanism. To understand the process of epigenetic inheritance, or propagation of histones and histone variants, as nucleosomes are disassembled and reassembled in living eukaryotic cells, we have explored the feasibility of exploiting photo-activated localization microscopy (PALM). PALM of single molecules in living cells has the potential to reveal new concepts in cell biology, providing insights into stochastic variation in cellular states. However, thus far, its use has been limited to studies in bacteria or to processes occurring near the surface of eukaryotic cells. With PALM, one literally observes and ‘counts’ individual molecules in cells one-by-one and this allows the recording of images with a resolution higher than that determined by the diffraction of light (the so-called super-resolution microscopy). Here, we investigate the use of different fluorophores and develop procedures to count the centromere-specific histone H3 variant CENP-ACnp1 with single-molecule sensitivity in fission yeast (Schizosaccharomyces pombe). The results obtained are validated by and compared with ChIP-seq analyses. Using this approach, CENP-ACnp1 levels at fission yeast (S. pombe) centromeres were followed as they change during the cell cycle. Our measurements show that CENP-ACnp1 is deposited solely during the G2 phase of the cell cycle.

Super-resolution microscopy is the term commonly given to fluorescence microscopy techniques with resolutions that are not limited by the diffraction of light. Since their conception a little over a decade ago, these techniques have quickly become the method of choice for many biologists studying structures and processes of single cells at the nanoscale. In this review, we present the three main approaches used to tackle the diffraction barrier of ∼200 nm: stimulated-emission depletion (STED) microscopy, structured illumination microscopy (SIM), and single-molecule localization microscopy (SMLM). We first present a theoretical overview of the techniques and underlying physics, followed by a practical guide to all of the facets involved in designing a super-resolution experiment, including an approachable explanation of the photochemistry involved, labeling methods available, and sample preparation procedures. Finally, we highlight some of the most exciting recent applications of and developments in these techniques, and discuss the outlook for this field.Keywords Super-resolution microscopy . Photophysics and photochemistry of fluorophores . Live cell imaging . Quantitative cell biology Spatial and temporal scales in the life sciences and microscopyThe timescales and spatial scales of the processes and molecules associated with life span extremely broad ranges, covering many orders of magnitude (Fig. 1). For instance, intracellular regulation (e.g., conformational changes or biochemical reactions within molecules) takes place at submillisecond timescales, nanosized molecules such as ATP (which serves the energy demands of cells) diffuse in milliseconds through cell volumes ranging from several micrometers up to millimeters, while (clustered) membrane receptors move at speeds that are about a magnitude slower. Large multicomponent machineries realize and control complex multilayered cellular functions that occur in seconds to hours. The ribosome, a large macromolecule which consists of two functional subunits of several dozen proteins on nucleic acid chain scaffolds, takes a matter of seconds to synthesize new peptide chains comprising hundreds of amino acids, which then quickly fold up into functional proteins. On the other hand, the replication of a full genome requires at least about 40 min for the 4.6 million nucleic acid base pairs of the bacterium Escherichia coli, and the cellular division cycle ranges from tens of minutes for E. coli to several hours for mammalian cells.Observing and understanding all of these components of life requires us to be, at best, passive witnesses of undisturbed processes, but also to demand hard observational data that can allow us to quantitatively measure and trace all of the players involved-ranging from small molecules up to the interactions of whole cells in cellular communities-with the highest specificity and precision.To achieve this, instrumentation is needed that permits a wide three-dimensional view but also allows details to be

We introduce a general approach for multicolor subdiffraction-resolution fluorescence imaging based on photoswitching of standard organic fluorophores. Photoswitching of ordinary fluorophores such as ATTO520, ATTO565, ATTO655, ATTO680, or ATTO700, i.e. the reversible transition from a fluorescent to a nonfluorescent state in aqueous buffers exploits the formation of long-lived triplet radical anions through reaction with reducing agents such as beta-mercaptoethylamine and repopulation of the singlet ground state by interaction with molecular oxygen. Thus, the time the different fluorophores reside in the fluorescent state can be easily adjusted by the excitation intensity and the concentration of the reducing agent. We demonstrate the potential of multicolor photoswitching microscopy with subdiffraction-resolution on cytoskeletal networks and molecular quantification of proteins in the inner mitochondrial membrane with approximately 20 nm optical resolution.