We describe a new concept of multimodal super-resolution imaging which combines the cumulant analysis from Super-resolution Optical Fluctuation Imaging (SOFT) with the imprinting of three-dimensional, spectral or other information into peculiar Point-Spread Function (PSF) patterns. This concept allows for encoding multidimensional or multimodal information into a single image plane and to extract this information by an appropriate spatio-temporal correlation analysis of emitter fluctuations. Here, we develop the general theory of this concept, and present proof-of-principle experiments of three-dimensional super-resolution imaging. Copyright (C) EPLA, 2019

This chapter focuses on two advanced fluorescence correlation spectroscopy (FCS) methods; fluorescence lifetime correlation spectroscopy (FLCS) and dual-focus FCS (2fFCS). We decided to put our focus on a detailed discussion of these two - and in our eyes well-merited - advanced methods, rather than giving an overview over the broad variety of advanced FCS methods that would consequently lack detail and leave the reader rather uneducated on all these methods. For this reason we had to exclude some candidates that would very well deserve the same amount of attention as the methods that we chose to focus on. Amongst these candidates camera-FCS, Bayes-FCS, and scanning-FCS are to be kept on the radar for sure.The great benefit of FLCS is that it provides a general tool that allows filtering for sub-populations, afterpulsing-artifacts, background effects, and basically anything that can be distinguished by its fluorescence lifetime. Complementarily, 2fFCS has brought a new level of accuracy to the table that has been previously reached only by complementary methods such as for example pulsed-field gradient NMR.

This volume focuses on Time-Correlated Single Photon Counting (TCSPC), a powerful tool allowing luminescence lifetime measurements to be made with high temporal resolution, even on single molecules. C

With the advent of superresolution imaging methods, fast dynamic imaging of biological processes in live cells remains a challenge. A subset of these methods requires the cellular targets to be labeled with spontaneously blinking probes. The delivery and specific targeting of cytosolic targets and the control of the probes' blinking properties are reviewed for three types of blinking probes: quantum dots, synthetic dyes, and fluorescent proteins.

We review the concept of superresolution optical fluctuation imaging (SOFI), discuss its attributes and trade-offs (in comparison with other superresolution methods), and present superresolved images taken on samples stained with quantum dots, organic dyes, and plasmonic metal nanoparticles. We also discuss the prospects of SOFI for live cell superresolution imaging and for imaging with other (non-fluorescent) contrasts.

Resolving fine details of subcellular structures is key to understanding the organization and function of cellular networks. Recent advances in far-field fluorescence microscopy provide the necessary tools to analyze these structures with resolutions well below the classical diffraction limit in all three dimensions. Technical improvements go hand-in-hand with new versions of switchable fluorophores that allow nonlinear optical effects to be more efficiently used to push the resolution limit down further. High contrast combined with the wide spectrum of available colors currently endow these fluorescencebased super-resolution techniques with the power to study the complexity of subcellular organelles and the relation of their constituting components down to the molecular level and under physiological conditions. In this way, they give us a far better understanding of the assembly of macro molecular complexes and their functions within a cell than has been possible before employing conventional imaging methods. In this review, we give an overview of the technical state-of-the art of these technologies, their fundamental and technical trade-offs, and provide typical application examples in this exciting field.

The past decade has seen an explosive growth in the utilization of single-molecule techniques for the study of complex systems. The ability to resolve phenomena otherwise masked by ensemble averaging has made these approaches especially attractive for the study of biological systems, where stochastic events lead to inherent inhomogeneity at the population level. The complex composition of the genome has made it an ideal system to study at the single-molecule level, and methods aimed at resolving genetic information from long, individual, genomic DNA molecules have been in use for the last 30 years. These methods, and particularly optical-based mapping of DNA, have been instrumental in highlighting genomic variation and contributed significantly to the assembly of many genomes including the human genome. Nanotechnology and nanoscopy have been a strong driving force for advancing genomic mapping approaches, allowing both better manipulation of DNA on the nanoscale and enhanced optical resolving power for analysis of genomic information. During the past few years, these developments have been adopted also for epigenetic studies. The common principle for these studies is the use of advanced optical microscopy for the detection of fluorescently labeled epigenetic marks on long, extended DNA molecules. Here we will discuss recent single-molecule studies for the mapping of chromatin composition and epigenetic DNA modifications, such as DNA methylation.

Multi-color super-resolution (SR) imaging microscopy techniques can resolve ultrastructural relationships between- and provide co-localization information of- different proteins inside the cell or even within organelles at a higher resolution than afforded by conventional diffraction-limited imaging. While still very challenging, important SR colocalization results have been reported in recent years using STED, PALM and STORM techniques.

Das Genom des Bakteriophagen T7 kann mithilfe einer sequenzspezifischen, Methyltransferase-induzierten DNA-Markierung durchmustert werden. In der Zuschrift auf S. 3638 zeigen E. Weinhold, S. Weiss, Y. Ebenstein et al., wie die Positionen von RNA-Polymerasen, die an DNA gebunden sind, als linearer optischer Strichcode verbildlicht werden können. Dadurch werden Strukturvariationen der genomischen DNA analysierbar, und zusätzliche Informationen über das Genom werden auf Einzelmolekülebene zugänglich.

The genome of the T7 bacteriophage can be mapped by using sequence-specific methyltransferase-induced labeling of DNA. In their Communication on page 3578 ff., E. Weinhold, S. Weiss, Y. Ebenstein, and co-workers show that the location of RNA polymerases that are bound to DNA can be visualized as a linear optical barcode, which allows structural variations in genomic DNA to be analyzed and provides an extra layer of contextual information about the genome at the single-molecule level.

Affordable DNA sequencing is revolutionizing genetic research and is enabling multiple novel biomedical applications. Among the inherent properties of today’s high-throughput sequencing technologies is the fact that it compiles long-range sequences from the assembly of numerous short-read data.[1] This leads to two fundamental limitations: loss of long-range contextual information on the single-genome level and difficulties coping with repetitive or variable genomic regions. Optical mapping and its variants[2–10] rely on the visualization of individual, long (50 kb–1000 kb) DNA molecules and extraction of genomic information by fluorescent labeling of the DNA. These techniques lack the resolution of sequencing but offer genomic context and therefore are attractive both in combination with sequencing to aid in sequence assembly[11–13] and for investigation of genomic structural variations on the individual chromosome level.[14, 15] Such variations include deletions, duplications, copy-number variants (CNVs), insertions, inversions, and translocations, all of which have a major impact on the phenotypic variations within a population (or somatic mutations, important in cancer progression). In addition, the available information content of the genome extends beyond the sequence, and the long-range data offered by optical mapping may provide crucial information regarding the distribution of DNA-binding proteins such as transcription factors and histones along the genome.

Superresolution microscopy has shifted the limits for fluorescence microscopy in cell -biology. The possibility to image cellular structures and dynamics of fixed and even live cells and organisms at resolutions of several nanometers holds great promise for future biological discoveries. We recently introduced a novel superresolution technique, based on the statistical evaluation of stochastic fluctuations stemming from single emitters, dubbed "superresolution optical fluctuation -imaging" (SOFI). In comparison to previously introduced superresolution methods, SOFI exhibits favorable attributes such as simplicity, affordability, high speed, and low levels of light exposure. Here we summarize the basic working principle and recent advances.

BACKGROUND:Fluorescence-based biological imaging has been revolutionized by the recent introduction of superresolution microscopy methods. 3D superresolution microscopy, however, remains a challenge as its implementation by existing superresolution methods is non-trivial.METHODS:Here we demonstrate a facile and straightforward 3D superresolution imaging and sectioning of the cytoskeletal network of a fixed cell using superresolution optical fluctuation imaging (SOFI) performed on a conventional lamp-based widefield microscope.RESULTS AND CONCLUSION:SOFI's inherent sectioning capability effectively transforms a conventional widefield microscope into a superresolution 'confocal widefield' microscope.

SOFI steht für „Superresolution Optical Fluctuation Imaging“, die jüngste einer Reihe sehr hoch auflösender Weitfeldmikroskopie-Techniken. Das Verfahren benötigt nur wenig Aufnahmezeit und lässt sich auf Proben anwenden, die mit herkömmlichen organischen Farbstoffen markiert sind. Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Superresolution Optical Fluctuation Imaging (SOFI) as initially demonstrated allows for a resolution enhancement in imaging by a factor of square-root of two. Here, we demonstrate how to increase the resolution of SOFI images by re-weighting the Optical Transfer Function (OTF). Furthermore, we demonstrate how cross-cumulants can be exploited to obtain a fair approximation of the underlying Point-Spread Function. We show a two-fold increase of resolution (over the diffraction limit) of near-infrared quantum dot labeled tubulin-network of 3T3 fibroblasts.

Super-resolution optical microscopy is a rapidly evolving area of fluorescence microscopy with a tremendous potential for impacting many fields of science. Several super-resolution methods have been developed over the last decade, all capable of overcoming the fundamental diffraction limit of light. We present here an approach for obtaining subdiffraction limit optical resolution in all three dimensions. This method relies on higher-order statistical analysis of temporal fluctuations (caused by fluorescence blinking/intermittency) recorded in a sequence of images (movie). We demonstrate a 5-fold improvement in spatial resolution by using a conventional wide-field microscope. This resolution enhancement is achieved in iterative discrete steps, which in turn allows the evaluation of images at different resolution levels. Even at the lowest level of resolution enhancement, our method features significant background reduction and thus contrast enhancement and is demonstrated on quantum dot-labeled microtubules of fibroblast cells.

In this report we evaluate the emission properties of single quantum dots embedded in a thin, thiol-containing polymer film. We report the suppression of quantum dot blinking leading to a continuous photon flux from both organic and water soluble quantum dots and demonstrate their application as robust fluorescent point sources for ultrahigh resolution localization. In addition, we apply the polymer coating to cell samples immunostained with antibody conjugated QDs and show that fluorescence intensity from the polymer embedded cells shows no sign of degradation after 67 h of continuous excitation. The reported thin polymer film coating may prove advantageous for immuno-cyto/histo-chemistry as well as for the fabrication of quantum dot containing devices requiring a reliable and stable photon source (including a single photon source) or stable charge characteristics while maintaining intimate contact between the quantum dot and the surrounding matrix.

Fluorescence correlation spectroscopy (FCS) is an important spectroscopic technique which can be used for measuring the diffusion and thus size of fluorescing molecules at pico- to nanomolar concentrations. Recently, we introduced an extension of conventional FCS, which is called dual-focus FCS (2fFCS) and allows absolute diffusion measurements with high precision and repeatability. It was shown experimentally that the method is robust against most optical and sample artefacts which are troubling conventional FCS measurements, and is furthermore able to yield absolute values of diffusion coefficients without referencing against known standards. However, a thorough theoretical treatment of the performance of 2fFCS is still missing. The present paper aims at filling this gap. Here, we have systematically studied the performance of 2fFCS with respect to the most important optical and photophysical factors such as cover slide thick-ness, refractive index of the sample, laser beam geometry, and optical satu-ration. We show that 2fFCS has indeed a superior performance when com-pared with conventional FCS, being mostly insensitive to most potential ab-errations when working under optimized conditions.

Fluorescence Correlation Spectroscopy (FCS) has been invented more than 30 years ago and experienced a renaissance after stable and affordable laser sources and low-noise single-photon detectors have become available. Its ability to measure diffusion coefficients at nanomolar concentrations of analyte made it a widely used tool in biophysics. However, in recent years it has been shown by many authors that aberrational (e.g. astigmatism) and photophysical effects (e.g. optical saturation) may influence the result of an FCS experiment dramatically, so that a precise and reliable estimation of the diffusion coefficient is no longer possible. Here, we report on the development, implementation, and application of a new and robust modification of FCS that we termed two-focus FCS (2fFCS) and which fulfils two requirements: (i) It introduces an external ruler into the measurement by generating two overlapping laser foci of precisely known and fixed distance. (ii) These two foci and corresponding detection regions are generated in such a way that the corresponding molecule detection functions (MDFs) are sufficiently well described by a simple two-parameter model yielding accurate diffusion coefficients when applied to 2fFCS data analysis. Both these properties enable us to measure absolute values of the diffusion coefficient with an accuracy of a few percent. Moreover, it turns out that the new technique is robust against refractive index mismatch, coverslide thickness deviations, and optical saturation effects, which so often trouble conventional FCS measurements. Additionally, we will show data that indicates that with 2fFCS it is even possible to monitor conformational changes of a calcium bindig protein affecting the hydrodynamic radius by as little as two Angstrom.