Advances in superresolution optical fluctuation imaging (SOFI).

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.

Widely accessible method for superresolution fluorescence imaging of living systems.

Superresolution fluorescence microscopy overcomes the diffraction resolution barrier and allows the molecular intricacies of life to be revealed with greatly enhanced detail. However, many current superresolution techniques still face limitations and their implementation is typically associated with a steep learning curve. Patterned illumination-based superresolution techniques [e.g., stimulated emission depletion (STED), reversible optically-linear fluorescence transitions (RESOLFT), and saturated structured illumination microscopy (SSIM)] require specialized equipment, whereas single-molecule-based approaches [e.g., stochastic optical reconstruction microscopy (STORM), photo-activation localization microscopy (PALM), and fluorescence-PALM (F-PALM)] involve repetitive single-molecule localization, which requires its own set of expertise and is also temporally demanding. Here we present a superresolution fluorescence imaging method, photochromic stochastic optical fluctuation imaging (pcSOFI). In this method, irradiating a reversibly photoswitching fluorescent protein at an appropriate wavelength produces robust single-molecule intensity fluctuations, from which a superresolution picture can be extracted by a statistical analysis of the fluctuations in each pixel as a function of time, as previously demonstrated in SOFI. This method, which uses off-the-shelf equipment, genetically encodable labels, and simple and rapid data acquisition, is capable of providing two- to threefold-enhanced spatial resolution, significant background rejection, markedly improved contrast, and favorable temporal resolution in living cells. Furthermore, both 3D and multicolor imaging are readily achievable. Because of its ease of use and high performance, we anticipate that pcSOFI will prove an attractive approach for superresolution imaging.

Superresolution optical fluctuation imaging (SOFI).

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.

Achieving increased resolution and more pixels with Superresolution Optical Fluctuation Imaging (SOFI).

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.

Measuring diffusion with polarization-modulation dual-focus fluorescence correlation spectroscopy.

We present a new technique, polarization-modulation dual-focus fluorescence correlation spectroscopy (pmFCS), based on the recently intro-duced dual-focus fluorescence correlation spectroscopy (2fFCS) to measure the absolute value of diffusion coefficients of fluorescent molecules at pico- to nanomolar concentrations. Analogous to 2fFCS, the new technique is robust against optical saturation in yielding correct values of the diffusion coefficient. This is in stark contrast to conventional FCS where optical saturation leads to an apparent decrease in the determined diffusion coefficient with increasing excitation power. However, compared to 2fFCS, the new technique is simpler to implement into a conventional confocal microscope setup and is compatible with cw-excitation, only needing as add-ons an electro-optical modulator and a differential interference contrast prism. With pmFCS, the measured diffusion coefficient (D) for Atto655 maleimide in water at 25?C is determined to be equal to (4.09 +/- 0.07) x 10(-6)cm(2)/s, in good agreement with the value of 4.04 x 10-6cm2/s as measured by 2fFCS.

The optics and performance of dual-focus fluorescence correlation spectroscopy.

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.

Toward single-molecule optical mapping of the epigenome.

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.

Labeling Cytosolic Targets in Live Cells with Blinking Probes.

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.

Resolving the spatial relationship between intracellular components by dual color super resolution optical fluctuations imaging (SOFI).

BACKGROUND: Multi-color super-resolution (SR) imaging microscopy techniques can resolve ultrastructura 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. RESULTS: In this work, we demonstrate dual-color Super Resolution Optical Fluctuations Imaging (SOFI) using a standard far-field fluorescence microscope and different color blinking quantum dots. We define the spatial relationship between hDcp1a, a processing body (P-body, PB) protein, and the tubulin cytoskeletal network. Our finding could open up new perspectives on the role of the cytoskeleton in PB formation and assembly. Further insights into PB internal organization are also reported and discussed. CONCLUSIONS: Our results demonstrate the suitability and facile use of multi-color SOFI for the investigation of intracellular ultrastructures.

SOFI-based 3D superresolution sectioning with a widefield microscope.

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.

Suppression of quantum dot blinking in DTT-doped polymer films.

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 hours 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.

Two-focus fluorescence correlation spectroscopy: a new tool for accurate and absolute diffusion measurements.

We present a new method to measure absolute diffusion coefficients at nanomolar concentrations with high precision. Based on a modified fluorescence correlation spectroscopy (FCS)-setup, this method is improved by introducing an external ruler for measuring the diffusion time by generating two laterally shifted and overlapping laser foci at a fixed and known distance. Data fitting is facilitated by a new two-parameter model to describe the molecule detection function (MDF). We present a recorded MDF and show the excellent agreement with the fitting model. We measure the diffusion coefficient of the red fluorescent dye Atto655 under various conditions and compare these values with a value achieved by gradient pulsed field NMR (GPF NMR). From these measurements we conclude, that the new measurement scheme is robust against optical and photophysical artefacts which are inherent to standard FCS. With two-focus-FCS, the diffusion coefficient of 4.26 x 10(-6) cm2s(-1) for Atto655 in water at 25 degrees C compares well with the GPF NMR value of 4.28 x 10(-6) cm2s(-1).

Surface sticking and lateral diffusion of lipids in supported bilayers.

The diffusion of fluorescently labeled lipids in supported bilayers is studied using two different methods: Z-scan fluorescence correlation spectroscopy (z-scan FCS) and two-focus fluorescence correlation spectroscopy (2f-FCS). It is found that the data can be fitted consistently only when taking into account partial sticking of the labeled lipids to the supporting glass surface. A kinetic reaction-diffusion model is developed and applied to the data. We find a very slow sticking rate which, however, when neglected, leads to strongly varying estimates of the free diffusion coefficient. The study reveals a strong sensitivity of FCS on even slight binding/unbinding kinetics of the labeled molecules, which has significance for related diffusion measurements in cellular lipid membranes.

Performance of fluorescence correlation spectroscopy for measuring diffusion and concentration.

Fluorescence correlation spectroscopy (FCS) has become an important tool for measuring diffusion, concentration, and molecular interactions of cellular components. The interpretation of FCS data critically depends on the measurement set-up. Here, we present a rigorous theory of FCS based on exact wave-optical calculations. Six of the most important optical and photophysical factors that influence FCS are studied: fluorescence anisotropy, cover-slide thickness, refractive index of the sample, laser-beam geometry, optical saturation, and pinhole adjustment. Our theoretical framework represents a general attempt to link all relevant parameters of the experimental set-up with the measured correlation function.