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La edad es un factor de riesgo arrítmico que, en especies animales, está asociado a la remodelación del colágeno. En este trabajo caracterizamos la dinámica tisular del colágeno asociada a la edad en el ventrículo izquierdo humano y utilizamos estos resultados para simular su efecto sobre la generación de arritmias ventriculares.

Cell–material interactions are crucial for many biomedical applications, including medical implants, tissue engineering, and biosensors. For implants, while the adhesion of eukaryotic host cells is desirable, bacterial adhesion often leads to infections. Surface free energy (SFE) is an important parameter that controls short- and long-term eukaryotic and prokaryotic cell adhesion. Understanding its effect at a fundamental level is essential for designing materials that minimize bacterial adhesion. Most cell adhesion studies for implants have focused on correlating surface wettability with mammalian cell adhesion and are restricted to short-term time scales. In this work, we used quartz crystal microbalance with dissipation monitoring (QCM-D) and electrical impedance analysis to characterize the adhesion and detachment of S. cerevisiae and E. coli, serving as model eukaryotic and prokaryotic cells within extended time scales. Measurements were performed on surfaces displaying different surface energies (Au, SiO2, and silanized SiO2). Our results demonstrate that tuning the surface free energy of materials is a useful strategy for selectively promoting eukaryotic cell adhesion and preventing bacterial adhesion. Specifically, we show that under flow and steady-state conditions and within time scales up to ∼10 h, a high SFE, especially its polar component, enhances S. cerevisiae adhesion and hinders E. coli adhesion. In the long term, however, both cells tend to detach, but less detachment occurs on surfaces with a high dispersive SFE contribution. The conclusions on S. cerevisiae are also valid for a second eukaryotic cell type, being the human embryonic kidney (HEK) cells on which we performed the same analysis for comparison. Furthermore, each cell adhesion phase is associated with unique cytoskeletal viscoelastic states, which are cell-type-specific and surface free energy-dependent and provide insights into the underlying adhesion mechanisms., info:eu-repo/semantics/published

To obtain accurate, reproducible, and interpretable data when conducting imaging experiments, it is critical to consider external factors affecting data acquisition at various steps of the experimental workflow. Illumination power and stability represent two critical factors, especially when comparing fluorescence intensities between images during a time-lapse experiment or experiments performed at different times or on other microscopes. The fluorescence signal can be generated by different types of light sources. These light sources and their coupling elements (e.g., fibers) can display varying performances over time as they age, move, or as environmental conditions change. Unfortunately, microscope users can often only set illumination power as a percentage of its maximal output and, may therefore, not be aware of potential performance changes. It is important to recognize that a set percentage will not always yield the same illumination power in Watts at the objective over the course of an experiment, not to mention between days or systems. This means that selecting for example 10% output may lead to different experimental results over time and will not necessarily be comparable to outputs obtained from other lasers or microscopes, even those of the very same model. If you are responsible for system maintenance, routinely measuring the illumination power, stability, and linearity over time can help you detect issues that affect the integrity of the system and thus the reproducibility of an experiment. This protocol describes how to measure the stability and linearity of the illumination power using calibrated external power sensors. This protocol is at the moment intended for confocal systems (raster scanning and spinning disks), but will be extended later to other imaging modalities. It represents the collective experience of 60 imaging scientists. Measurements made by our working group with this protocol are available in a public database, which will be updated with further contributions from the community.

Multicolor fluorescence imaging is an excellent method for the simultaneous visualization of multiple structures, although it is limited by the available spectral window. More labels can be measured by distinguishing these on properties, such as their fluorescence dynamics, but usually these dynamics must be directly resolvable by the instrument. We propose an approach to distinguish emitters over a much broader range of light-induced dynamics by combining fast modulation of the light source with the detection of the time-integrated fluorescence. We demonstrate our method by distinguishing four spectrally overlapping photochromic fluorophores within Escherichia coli bacteria, showing that we can accurately classify all four probes by acquiring just two to four fluorescence images. Our strategy expands the range of probes and processes that can be used for fluorescence multiplexing. ispartof: Biophysical Reports vol:1 issue:2 pages:100026- ispartof: location:United States status: published

Fluorescence microscopy is a standard research tool in many fields, although collecting reliable images can be difficult in systems characterized by low expression levels and/or high background fluorescence. We present the combination of a photochromic fluorescent protein and stochastic optical fluctuation imaging (SOFI) to deliver suppression of the background fluorescence. This strategy makes it possible to resolve lowly or endogenously expressed proteins, as we demonstrate for Gcn5, a histone acetyltransferase required for complete virulence, and Erg11, the target of the azole antifungal agents in the fungal pathogen Candida albicans. We expect that our method can be readily used for sensitive fluorescence measurements in systems characterized by high background fluorescence. IMPORTANCE Understanding the spatial and temporal organization of proteins of interest is key to unraveling cellular processes and identifying novel possible antifungal targets. Only a few therapeutic targets have been discovered in Candida albicans, and resistance mechanisms against these therapeutic agents are rapidly acquired. Fluorescence microscopy is a valuable tool to investigate molecular processes and assess the localization of possible antifungal targets. Unfortunately, fluorescence microscopy of C. albicans suffers from extensive autofluorescence. In this work, we present the use of a photochromic fluorescent protein and stochastic optical fluctua-tion imaging to enable the imaging of lowly expressed proteins in C. albicans through the suppression of autofluorescence. This method can be applied in C. albicans research or adapted for other fungal systems, allowing the visualization of intricate processes. Superscript/Subscript Available

Anisotropic environments can drastically alter the spectroscopy and photochemistry of molecules, leading to complex structure-function relationships. We examined this using fluorescent proteins as easy-to-modify model systems. Starting from a single scaffold, we have developed a range of 27 photochromic fluorescent proteins that cover a broad range of spectroscopic properties, including the determination of 43 crystal structures. Correlation and principal component analysis confirmed the complex relationship between structure and spectroscopy, but also allowed us to identify consistent trends and to relate these to the spatial organization. We find that changes in spectroscopic properties can come about through multiple underlying mechanisms, of which polarity, hydrogen bonding and presence of water molecules are key modulators. We anticipate that our findings and rich structure/spectroscopy dataset can open opportunities for the development and evaluation of new and existing protein engineering methods. We are grateful to Gerrit Groenhof (University of Jyvaskyla), Jeremy Harvey (KU Leuven), Raffaele Vitale (Universite de Lille), and Dominique Bourgeois (Institut de Biologie Structurale) for critical insights and discussion. E.D.Z. and L.V.M. thank the beamline staff from X06DA at the Swiss Light Source (Villigen, Switzerland), Proxima1 and Proxima2A at synchrotron Soleil (Gif-sur-Yvette, France), XRD1 at Elettra (Trieste, Italy) and I03 at Diamond Light Source (Oxfordshire, UK) for assistance during X-ray diffraction data collection. E.D.Z., S.H., and S.D. thank the Research Foundation Flanders (FWO) for a doctoral fellowship and postdoctoral fellowships (12X7919N and 12R2817N). This work was supported through funding from the Research Foundation Flanders through grants 1514319 N, G090819N, G0B8817N, and the European Research Council through grant 714688 NanoCellActivity. Dedecker, P (corresponding author), Katholieke Univ Leuven, Dept Chem, Celestijnenlaan 200G Box 2403, B-3001 Leuven, Belgium. peter.dedecker@kuleuven.be

Photochromic fluorescent proteins have become versatile tools in the life sciences, though our understanding of their structure-function relation is limited. Starting from a single scaffold, we have developed a range of 27 photochromic fluorescent proteins that cover a broad range of spectroscopic properties, yet differ only in one or two mutations. We also determined 43 different crystal structures of these mutants. Correlation and principal component analysis of the spectroscopic and structural properties confirmed the complex relationship between structure and spectroscopy, suggesting that the observed variability does not arise from a limited number of mechanisms, but also allowed us to identify consistent trends and to relate these to the spatial organization around the chromophore. We find that particular changes in spectroscopic properties can come about through multiple different underlying mechanisms, of which the polarity of the chromophore environment and hydrogen bonding of the chromophore are key modulators. Furthermore, some spectroscopic parameters, such as the photochromism, appear to be largely determined by a single or a few structural properties, while other parameters, such as the absorption maximum, do not allow a clear identification of a single cause. We also highlight the role of water molecules close to the chromophore in influencing photochromism. We anticipate that our dataset can open opportunities for the development and evaluation of new and existing protein engineering methods.

Microtubule-interacting drugs, sometimes referred to as antimitotics, are used in cancer therapy to target and disrupt micro-tubules. However, their side effects require the development of safer drug regimens that still retain clinical efficacy. Currently, many questions remain regarding microtubule-interacting drugs at clinically relevant and ultra-low doses. Here, we use super-resolution microscopies (single molecule localization and optical fluctuation based) to reveal the initial microtubule dysfunctions caused by nanomolar concentrations of colcemid. Short exposure to 30 - 80 nM colcemid results in aberrant microtubule curvature while microtubule fragmentation is detected upon treatment with ≥100 nM colcemid. Remarkably, even ultra-low doses (5 hours at

We report on a novel biomimetic sensor that allows sensitive and specific detection of Escherichia coli (E. coli) bacteria in a broad concentration range from 102 up to 106 CFU/mL in both buffer fluids and relevant food samples (i.e. apple juice). The receptors are surface-imprinted polyurethane layers deposited on stainless-steel chips. Regarding the transducer principle, the sensor measures the increase in thermal resistance between the chip and the liquid due to the presence of bacteria captured on the receptor surface. The low noise level that enables the low detection limit originates from a planar meander element that serves as both a heater and a temperature sensor. Furthermore, the experiments show that the presence of bacteria in a liquid enhances the thermal conductivity of the liquid itself. Reference tests with a set of other representative species of Enterobacteriaceae, closely related to E. coli, indicate a very low cross-sensitivity with a sensor response at or below the noise level.

Super-resolution optical fluctuation imaging (SOFI) provides super-resolution (SR) fluorescence imaging by analyzing fluctuations in the fluorophore emission. The technique has been used both to acquire quantitative SR images and to provide SR biosensing by monitoring changes in fluorophore blinking dynamics. Proper analysis of such data relies on a fully quantitative model of the imaging. However, previous SOFI imaging models made several assumptions that can not be realized in practice. In this work we address these limitations by developing and verifying a fully quantitative model that better approximates real-world imaging conditions. Our model shows that (i) SOFI images are free of bias, or can be made so, if the signal is stationary and fluorophores blink independently, (ii) allows a fully quantitative description of the link between SOFI imaging and probe dynamics, and (iii) paves the way for more advanced SOFI image reconstruction by offering a computationally fast way to calculate SOFI images for arbitrary probe, sample and instrumental properties. ispartof: Optics Express vol:27 issue:18 pages:25749-25766 ispartof: location:United States status: published

The field of super-resolution microscopy continues to progress rapidly, both in terms of evolving techniques and methodologies as well as in the development of new multi-disciplinary applications. Two current drivers of innovation are increasing the possible resolution gain and application in live samples. Super-resolution optical fluctuation imaging (SOFI) is well suited to live samples while expansion microscopy (ExM) enables obtainment of sub-diffraction information via conventional imaging. In this Highlight we provide a brief outline of these methods and report results from application of SOFI and ExM in our on-going study into microtubule remodelling by rabies virus P proteins. We show that MT bundles in live cells transfected with rabies virus P3 protein can be visualised using SOFI in a time-lapse fashion for up to half an hour and can be expanded using current Pro-ExM protocols and imaged using conventional microscopy.

The field of super-resolution microscopy continues to progress rapidly, both in terms of evolving techniques and methodologies as well as in the development of new multi-disciplinary applications. Two current drivers of innovation are increasing the possible resolution gain and application in live samples. Super-resolution optical fluctuation imaging (SOFI) is well suited to live samples while expansion microscopy (ExM) enables obtainment of sub-diffraction information via conventional imaging. In this Highlight we provide a brief outline of these methods and report results from application of SOFI and ExM in our on-going study into microtubule remodelling by rabies virus P proteins. We show that MT bundles in live cells transfected with rabies virus P3 protein can be visualised using SOFI in a time-lapse fashion for up to half an hour and can be expanded using current Pro-ExM protocols and imaged using conventional microscopy.

Super-resolution (SR) fluorescence microscopy is typically carried out on research microscopes equipped with high-NA TIRF objectives and powerful laser light sources. Super-resolution optical fluctuation imaging (SOFI) is a fast SR technique capable of live-cell imaging, that is compatible with many wide-field microscope systems. However, especially when employing fluorescent proteins, a key part of the imaging system is a very sensitive and well calibrated camera sensor. The substantial costs of such systems preclude many research groups from employing SR imaging techniques. Here, we examine to what extent SOFI can be performed using a range of imaging hardware comprising different technologies and costs. In particular, we quantitatively compare the performance of an industry-grade CMOS camera to both state-of-the-art emCCD and sCMOS detectors, with SOFI-specific metrics. We show that SOFI data can be obtained using a cost-efficient industry-grade sensor, both on commercial and home-built microscope systems, though our analysis also readily exposes the merits of the per-pixel corrections performed in scientific cameras.

The complex microscopic nature of many live biological processes is often obscured by the diffraction limit of light, requiring diffraction-unlimited fluorescence microscopy to resolve them. Because of the vast range of different processes that can be studied, sub-diffraction imaging should work efficiently under many different conditions. Photochromic stochastic optical fluctuation imaging (pcSOFI) is a recent addition to the field of diffraction-unlimited fluorescence microscopy. This robust and versatile method employs a statistical analysis of random fluctuations in the emission of single labels, in this case reversibly switchable fluorescent proteins (RSFPs), to retrieve super-resolution information. Added to the resolution enhancement, pcSOFI also offers contrast enhancement and background reduction in a practical and convenient way. Here, we describe the necessary steps to obtain diffraction-unlimited images, including multicolor and three-dimensional imaging, and highlight the advantages of pcSOFI together with the circumstances under which pcSOFI can be favorably applied.

Super-resolution optical fluctuation imaging overcomes the diffraction limit by analyzing fluctuations in the fluorophore emission. A key assumption of the imaging is that the fluorophores are independent, though this is invalidated in the presence of photodestruction. In this work, we evaluate the effect of photodestruction on SOFI imaging using theoretical considerations and computer simulations. We find that photodestruction gives rise to an additional signal that does not present an easily interpretable view of the sample structure. This additional signal is strong and the resulting images typically exhibit less noise. Accordingly, these images may be mis-interpreted as being more visually pleasing or more informative. To address this uncertainty, we develop a procedure that can robustly estimate to what extent any particular experiment is affected by photodestruction. We also develop a detailed assessment methodology and use it to evaluate the performance of several correction algorithms. We identify two approaches that can correct for the presence of even strong photodestruction, one of which can be implemented directly in the SOFI calculation software. ispartof: SCIENTIFIC REPORTS vol:7 issue:1 ispartof: location:England status: published

Reversibly switchable fluorescent proteins (RSFPs) enable advanced fluorescence imaging, though the performance of this imaging crucially depends on the properties of the labels. We report on the use of an existing small binding peptide, named Enhancer, to modulate the spectroscopic properties of the recently developed rsGreen series of RSFPs. Fusion constructs of Enhancer with rsGreen1 and rsGreenF revealed an increased molecular brightness and pH stability, although expression in living E. coli or HeLa cells resulted in a decrease of the overall emission. Surprisingly, Enhancer binding also increased off-switching speed and resistance to switching fatigue. Further investigation suggested that the RSFPs can interconvert between fast- and slow-switching emissive states, with the overall protein population gradually converting to the slow-switching state through irradiation. The Enhancer modulates the spectroscopic properties of both states, but also preferentially stabilizes the fast-switching state, supporting the increased fatigue resistance. This work demonstrates how the photo-physical properties of RSFPs can be influenced by their binding to other small proteins, which opens up new horizons for applications that may require such modulation. Furthermore, we provide new insights into the photoswitching kinetics that should be of general consideration when developing new RSFPs with improved or different photochromic properties. ispartof: International Journal Of Molecular Sciences vol:18 issue:9 ispartof: location:Switzerland status: published

Scientific Reports 6: Article number: 21413; Published online 25 February 2016; Updated: 09 May 2016 A marked-up version of the Supplementary Information was inadvertently published with the original version of this Article. In addition, Supplementary Software 1 was omitted. These errors have now been corrected.

Stochastic optical fluctuation imaging (SOFI) is a super-resolution fluorescence imaging technique that makes use of stochastic fluctuations in the emission of the fluorophores. During a SOFI measurement multiple fluorescence images are acquired from the sample, followed by the calculation of the spatiotemporal cumulants of the intensities observed at each position. Compared to other techniques, SOFI works well under conditions of low signal-to-noise, high background, or high emitter densities. However, it can be difficult to unambiguously determine the reliability of images produced by any superresolution imaging technique. In this work we present a strategy that enables the estimation of the variance or uncertainty associated with each pixel in the SOFI image. In addition to estimating the image quality or reliability, we show that this can be used to optimize the signal-to-noise ratio (SNR) of SOFI images by including multiple pixel combinations in the cumulant calculation. We present an algorithm to perform this optimization, which automatically takes all relevant instrumental, sample, and probe parameters into account. Depending on the optical magnification of the system, this strategy can be used to improve the SNR of a SOFI image by 40% to 90%. This gain in information is entirely free, in the sense that it does not require additional efforts or complications. Alternatively our approach can be applied to reduce the number of fluorescence images to meet a particular quality level by about 30% to 50%, strongly improving the temporal resolution of SOFI imaging. ispartof: Biomedical Optics Express vol:7 issue:2 pages:467-480 ispartof: location:United States status: published

It has become increasingly clear that protein-protein interactions (PPIs) are compartmentalized in nano- scale domains that define the biochemical architec- ture of the cell. Despite tremendous advances in su- per-resolution imaging, strategies to observe PPIs at sufficient resolution to discern their organization are just emerging. Here we describe a strategy in which PPIs induce reconstitution of fluorescent proteins (FPs) that are capable of exhibiting single-molecule fluctuations suitable for stochastic optical fluctuation imaging (SOFI). Subsequently, spatial maps of these interactions can be resolved in super-resolution in living cells. Using this strategy, termed reconstituted fluorescence-based SOFI (refSOFI), we investigated the interaction between the endoplasmic reticulum (ER) Ca2+ sensor STIM1 and the pore-forming chan- nel subunit ORAI1, a crucial process in store-oper- ated Ca2+ entry (SOCE). Stimulating SOCE does not appear to change the size of existing STIM1/ORAI1 interaction puncta at the ER-plasmamembrane junc- tions, but results in an apparent increase in the num- ber of interaction puncta. publisher: Elsevier articletitle: RefSOFI for Mapping Nanoscale Organization of Protein-Protein Interactions in Living Cells journaltitle: Cell Reports articlelink: http://dx.doi.org/10.1016/j.celrep.2015.12.036 content_type: article copyright: Copyright © 2016 The Authors. Published by Elsevier Inc. ispartof: Cell Reports vol:14 issue:2 pages:390-400 ispartof: location:United States status: published

The complex microscopic nature of many live biological processes is often obscured by the diffraction limit of light, requiring diffraction-unlimited fluorescence microscopy to resolve them. Because of the vast range of different processes that can be studied, sub-diffraction imaging should work efficiently under many different conditions. Photochromic stochastic optical fluctuation imaging (pcSOFI) is a recent addition to the field of diffraction-unlimited fluorescence microscopy. This robust and versatile method employs a statistical analysis of random fluctuations in the emission of single labels, in this case reversibly switchable fluorescent proteins (RSFPs), to retrieve super-resolution information. Added to the resolution enhancement, pcSOFI also offers contrast enhancement and background reduction in a practical and convenient way. Here, we describe the necessary steps to obtain diffraction-unlimited images, including multicolor and three-dimensional imaging, and highlight the advantages of pcSOFI together with the circumstances under which pcSOFI can be favorably applied.

International audience; “Smart fluorophores”, such as reversibly switchable fluorescent proteins, are crucial for advanced fluorescence imaging. However, only a limited number of such labels is available, and many display reduced biological performance compared to more classical variants. We present the development of robustly photoswitchable variants of enhanced green fluorescent protein (EGFP), named rsGreens, that display up to 30-fold higher fluorescence in E. coli colonies grown at 37 °C and more than 4-fold higher fluorescence when expressed in HEK293T cells compared to their ancestor protein rsEGFP. This enhancement is not due to an intrinsic increase in the fluorescence brightness of the probes, but rather due to enhanced expression levels that allow many more probe molecules to be functional at any given time. We developed rsGreens displaying a range of photoswitching kinetics and show how these can be used for multimodal diffraction-unlimited fluorescence imaging such as pcSOFI and RESOLFT, achieving a spatial resolution of ∼70 nm. By determining the first ever crystal structures of a negative reversibly switchable FP derived from Aequorea victoria in both the “on”- and “off”-conformation we were able to confirm the presence of a cis–trans isomerization and provide further insights into the mechanisms underlying the photochromism. Our work demonstrates that genetically encoded “smart fluorophores” can be readily optimized for biological performance and provides a practical strategy for developing maturation- and stability-enhanced photochromic fluorescent proteins.

We present Localizer, a freely available and open source software package that implements the com- putational data processing inherent to several types of superresolution fluorescence imaging, such as localization (PALM/STORM/GSDIM) and fluctuation imaging (SOFI/pcSOFI). Localizer delivers high accuracy and performance and comes with a fully featured and easy-to-use graphical user interface but is also designed to be integrated in higher-level analysis environments. Due to its modular design, Localizer can be readily extended with new algo- rithms as they become available, while maintaining the same interface and performance. Weprovide front-ends for running Localizer from Igor Pro, Matlab, or as a stand-alone program. We show that Localizer performs favorably when compared with two existing superresolution packages, and to our knowledge is the only freely available implementation of SOFI/pcSOFI microscopy. By dramatically improving the analysis performance and ensuring the easy addition of current and future enhancements, Localizer strongly improves the usability of superresolution imaging in a variety of biomedical studies. ispartof: Journal of Biomedical Optics vol:17 issue:12 pages:1-5 ispartof: location:United States status: published

“Smart fluorophores”, such as reversibly switchable fluorescent proteins, are crucial for advanced fluorescence imaging. However, only a limited number of such labels is available, and many display reduced biological performance compared to more classical variants. We present the development of robustly photoswitchable variants of enhanced green fluorescent protein (EGFP), named rsGreens, that display up to 30-fold higher fluorescence in E. coli colonies grown at 37 °C and more than 4-fold higher fluorescence when expressed in HEK293T cells compared to their ancestor protein rsEGFP. This enhancement is not due to an intrinsic increase in the fluorescence brightness of the probes, but rather due to enhanced expression levels that allow many more probe molecules to be functional at any given time. We developed rsGreens displaying a range of photoswitching kinetics and show how these can be used for multimodal diffraction-unlimited fluorescence imaging such as pcSOFI and RESOLFT, achieving a spatial resolution of ∼70 nm. By determining the first ever crystal structures of a negative reversibly switchable FP derived from Aequorea victoria in both the “on”- and “off”-conformation we were able to confirm the presence of a cis-trans isomerization and provide further insights into the mechanisms underlying the photochromism. Our work demonstrates that genetically encoded “smart fluorophores” can be readily optimized for biological performance and provides a practical strategy for developing maturation- and stability-enhanced photochromic fluorescent proteins.

Fluorescent proteins (FPs) and fluorescent protein-derived biosensors are indispensable tools in life sciences. They make it possible to probe the location, activity, or interaction of molecules of interest from a subcellular to a multicellular scale. The desire for high-resolution and multidimensional information imposes continuously increasing demands on the performance of the employed probes leading to a continued need for optimization and integration of additional features, such as phototransformable behavior. This review highlights the latest advances in FP engineering, which increase throughput and tailor the improvements directly to the envisioned experiments. Additionally, we discuss recent alternative approaches to introduce or alter phototransformable behavior and describe selected applications of phototransformable behavior in biosensors. ispartof: CURRENT OPINION IN BIOTECHNOLOGY vol:58 pages:183-191 ispartof: location:England status: published

Advances in image scanning microscopy move super-resolution imaging deeper into tissues with faster visualization and finer details. ispartof: Nature Methods vol:14 issue:11 pages:1041-1044 ispartof: location:United States status: published

Virtually all cellular processes depend on networks of highly dynamic protein-protein interactions (PPIs). Hence, deciphering cellular functions at the molecular level requires investigating the spatiotemporal characteristics of PPIs precisely within the context of these signaling networks. However, there is accumulating evidence that protein complexes are often organized into microdomains or nanodomains whose size is below the diffraction limit and thus cannot be sufficiently characterized using standard imaging techniques. Despite the tremendous progress in establishing and refining super-resolution imaging techniques for fluorescently labeled proteins in live cells, observation of protein-protein interactions (PPIs) in super-resolution remains challenging. To address this problem, we developed a strategy in which PPIs induce reconstitution of different fluorescent proteins, whereupon these interactions are resolved in super-resolution using Stochastic Optical Fluctuation Imaging (SOFI) within 30 minutes after complex formation. We employed this reconstituted fluorescence based SOFI (refSOFI) to investigate the interaction between the endoplasmic reticulum Ca2+ sensor STIM1 and the pore forming channel subunit ORAI1, a process crucial for store-operated Ca2+ entry. We found that STIM1-ORAI1 interaction puncta exhibit a smaller size than that suggested by current fluorescence microscopy-based assessments. Moreover, we discovered that the density of these punctate structures is not necessarily dependent on their size. In fact, we observed that STIM1-ORAI1 activation predominantly leads to the formation of new, smaller puncta instead of larger complexes. However, we identified that other perturbing factors can significantly change the size of puncta. These insights critically depend on the obtained super-resolution information. In conclusion, refSOFI represents a versatile tool to examine PPIs in living cells in super-resolution.