Thomas Dertinger

Fast, background-free, 3D super-resolution optical fluctuation imaging (SOFI)

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.

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.

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

Superresolution Optical Fluctuation Imaging with Organic Dyes

Superresolution far-field microscopy has experienced a tremendous growth since the introduction of the first concept in 1994[1], broadening the scope and applications of fluorescence microscopy beyond the diffraction limit. Various powerful and excellent methods, such as stimulated emission depletion[2], saturated structured illumination microscopy[3] and stochastic single-molecule switching methods such as STORM[4], (f)PALM[5, 6], directSTORM (dSTORM)[7, 8] and GSDIM[9], have been developed since. Notably, the key to all superresolution methods lies in the exploitation of a two-state transition (e.g. a fluorescent ‘on’ and a non-fluorescent ‘off’ state) of the molecules. Especially for the stochastic single-molecule switching methods (PALM, STORM etc.) it is necessary to precisely control both the time a fluorophore spends in the fluorescent as well as in the dark state. In the dSTORM concept, this is achieved by irradiation of the sample with one or two wavelengths in the presence of reducing agents. In parallel, the invention of a reducing and oxidizing buffer system (ROXS) made the fluorescence intermittency rates of most organic dyes tuneable to almost any degree[10, 11]. Immediate consequences are increased photo-stability and ‘non-blinking’ fluorescent dyes, which has been recently exploited to increase the performance of STED[12]. Here we demonstrate that reversible photoswitching of organic dyes using similar conditions as in dSTORM can be utilized for another superresolution technique, namely Superresolution Optical Fluctuation Imaging (SOFI)[13]. SOFI makes use of random temporal signal fluctuations of single emitters and uses them to achieve fast, background-free, 3D superresolution microscopy by means of higher order statistics (HOS). In contrast to single-molecule switching methods, many emitters can be ‘on’ at the same time in a diffraction-limited volume and still contribute to enhanced resolution. The SOFI protocol consists of recording a movie of the fluctuating signal on any kind of imaging platform. This movie is then processed by a software-based HOS analysis (that could also be implemented in hardware). So far, SOFI has been demonstrated to work with blinking quantum dots (QDs) and a resolution enhancement of a factor of two could be achieved in generic imaging applications[14]. Since almost all organic dyes exhibit fluorescence intermittency (e.g. due to intersystem crossing to the dark triplet state, which gives rise to a fluctuating fluorescence signal), they could potentially be used for SOFI as well. The use of much smaller and targetable organic dyes (as compared to QDs) could expand the scope of SOFI to a large array of live and fixed cell imaging applications. On the other hand, using dyes for SOFI introduces many challenges: (i) the immanent photo-bleaching of dyes upon illumination limits the acquisition time and potentially compromises the SOFI algorithm (since it requires a temporally constant mean signal); (ii) a lower signal to noise ratio (as compared to QDs) degrades the algorithm’s performance; (iii) typical intersystem crossing rates of conventional organic dyes result in microseconds ‘on/off’ blinking, whereas most movie acquisitions for SOFI are limited to millisecond timescales. Here we apply experimental conditions similar to the ones used in the dSTORM concept to adjust reversible photoswitching of organic dyes to a suitable level for SOFI, using an EMCCD camera acquisition. We chose the dye Alexa647 (which exhibits light-driven microsecond fluorescence intermittency) for these experiments (see Experimental Section)[7]. Note that similar experimental conditions were shown to control blinking of a large selection of organic dyes[8] and therefore our choice of a cyanine dye is almost arbitrary. The experiment was carried out on fixed COS-7 cells whose β-tubulin network was immuno-labelled with Alexa647-conjugated antibodies. We recorded a movie of 1000 frames at a frame rate of 20 Hz (see Supporting Information) and subsequently analyzed it with an extended SOFI algorithm[14]. This enabled us to produce SOFI images that have four-times more pixels than the original acquired image. Fig. 1 clearly shows resolution enhancement, and in addition, a striking reduction in background fluorescence can be observed in the SOFI image. This is due to the fact that the SOFI algorithm inherently eliminates the non-fluctuating background signal. Also, the inherent SOFI’s optical sectioning attribute contributes to a ‘cleaner’ image since out-of-focus light is suppressed even though imaging was done in close-to-TIRF configuration. The resolution enhancement translates in a smaller Rayleigh limit of 169 nm (as compared to 290 nm for the conventional imaging), which was assessed by comparing the same line profile in Fig. 1a and 1b (Fig. 1c). Figure 1 Fluorescence and corresponding SOFI images of β-tubulin network of COS-7 cells. White boxes are magnified regions shown in the upper right corner. An intensity cross-section (white line) is taken to evaluate the resolution enhancement. (A) Original ... In order to circumvent imaging artefacts due to dye photo-bleaching during acquisition (which would manifest as non-resolved features in the SOFI image), the acquired movie was analysed piecewise in blocks of frames. Within each block, the change of the mean signal due to photo-bleaching was negligible (see Experimental Section) and therefore the requirement for successful SOFI imaging was given for each movie block. SOFI images for all movie blocks were summed together before any further analysis. In conclusion, we demonstrated a convenient superresolution imaging method using conventional organic dyes with an unmodified TIRF-microscope. Compared to single-molecule superresolution methods based on switching, SOFI reached superresolution performance in equivalent acquisition times (50 s for the data presented here). It has, however, the potential to run at even faster speeds (topic of current research). Note that even though we applied photoswitching conditions in order to tune the on/off times, the only SOFI prerequisite was to be able to monitor dye fluctuations. Therefore, it is likely that SOFI could be implemented in the future with faster blinking dyes as faster cameras come online[15, 16]. In contrast to PALM and STORM, SOFI does not require long off times or has to maintain a certain on/off time ratio. This obviously points to future experiments, which will be performed in live cells, where tuning capabilities and measurement times are limited and fast acquisition is crucial. Since the signal to noise ratio of dyes is considerably lower in comparison to QDs, SOFI imaging is more demanding, i.e. for QDs an arbitrary frame rate of the camera can be used (leading to an improved signal) whereas for dyes a fast frame rate is desirable since photo-bleaching limits the acquisition time. Photo-bleaching is also the limiting factor for applying higher-order correlations in order to improve resolution more than demonstrated here.

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.

Enzymatically Incorporated Genomic Tags for Optical Mapping of DNA-Binding Proteins

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.

SOFI-based 3D superresolution sectioning with a widefield microscope

BackgroundFluorescence-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.MethodsHere 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 ConclusionSOFI’s inherent sectioning capability effectively transforms a conventional widefield microscope into a superresolution ‘confocal widefield’ microscope.

Art and artifacts of fluorescence correlation spectroscopy

Fluorescence correlation spectroscopy (FCS) is an important technique for studying analyte molecules on a single molecule level in solution. The core molecular characteristic that is addressed by FCS is the translational diffusion coefficient of the analyte molecules, which can be used for studying molecular binding interactions or conformational changes of macromolecules. We present a thorough theoretical analysis of the FCS technique, paying special attention to the various frequently occurring technical artifacts. Particularly, we consider the influence of refractive index mismatch, cover-slide thickness, fluorescence anisotropy, optical adjustment, and optical saturation on the measured autocorrelation curve (ACF). The impact of these factors on the apparently determined diffusion coefficient is quantitatively evaluated. Extensive experimental results are presented demonstrating the theoretically predicted effects and dependencies.

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.

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.