@article {70266, title = {Mechanically-induced Septin Networks protect nuclear integrity}, journal = {bioRxiv}, year = {2026}, month = {2026 Jan 21}, abstract = {

The cytoskeleton is a key mediator of mechanical interactions in cells, but specific contributions of septins remains unclear. Septins preferentially localize with a subset of actin stress fibers positioned under the nucleus, where they are situated between the membrane and stress fibers. Removing the nucleus from the cell results in the loss of these subnuclear septin-decorated stress fibers. Surprisingly, however, their formation can be rescued using a large glass bead in place of the nucleus. Similarly, applying a compressive force to the cell via confinement, whether externally or through internally generated actomyosin forces, results in increased septin accumulation in regions where the nucleus engages the cell cortex. Finally, loss of septin filaments via knockdown of SEPT7 increases the likelihood of nuclear membrane rupture during confinement. Together these data suggest that septins act as a dynamic mechanosensitive protective mechanism to buffer mechanical forces on the nucleus.

}, doi = {10.64898/2026.01.20.700414}, url = {https://www.biorxiv.org/content/early/2026/01/21/2026.01.20.700414}, author = {Utgaard, Margaret E. and Caillier, Alexia and Chandrasekar, Shreya and Tidei, Joseph J. and Balasubramanian, Harikrushnan and Lee, Rachel M. and Puls, Owen and Khuon, Satya and Aaron, Jesse and Chew, Teng-Leong and Beach, Jordan R. and Oakes, Patrick W.} } @article {70294, title = {Phasor analysis of RGB camera data enables fluorescence microscopy unmixing and brightfield segmentation in a commercial microscope}, journal = {Sens Bio-Sens Res}, year = {2026}, month = {2026 Apr 18}, abstract = {

Spectral information plays a crucial role in biological imaging, yet conventional epifluorescence and histological techniques often rely on RGB image acquisition, limiting the resolution of spectrally overlapping components. Here, we present a phasor-based spectral analysis framework adapted for RGB images, enabling unsupervised segmentation and unmixing without the need for hyperspectral systems or sequential acquisition. By applying a discrete Fourier transform to the red, green, and blue intensities at each pixel, we generate a two-dimensional phasor plot where spectral relationships are encoded in modulation and phase. We demonstrate the utility of this approach across three distinct applications: segmentation of lung histology images stained with hematoxylin and eosin to quantify alveolar collapse, analysis of autofluorescence in skin lesions (nevi and melanoma) to highlight pathological spectral signatures, and spectral unmixing in multicolor-labeled U2OS cells to resolve overlapping fluorophores. Our method improves signal separation, reduces noise, and enhances biological interpretability using standard RGB acquisition. These findings establish RGB phasor analysis as a practical and powerful tool for spectral decomposition and segmentation in microscopy, bridging the gap between conventional imaging and advanced spectral analysis.

}, doi = {10.1016/j.sbsr.2026.101014}, url = {https://doi.org/10.1016/j.sbsr.2026.101014}, author = {Schuty, Bruno and Garcia, Maria Jose and Khuon, Satya and Malacrida, Leonel S} } @article {70257, title = {Redefining colocalization analysis with a novel phasor mixing coefficient}, journal = {Journal of Cell Science}, volume = {139}, year = {2026}, month = {2026 Jan 19}, pages = {jcs264388}, abstract = {

The first step to probing any potential interaction between two biomolecules is to determine their spatial association. In other words, if two biomolecules localize similarly within a cell, then it is plausible they could interact. Traditionally, this is quantified through various colocalization metrics. These measures infer this association by estimating the degree to which fluorescent signals from each biomolecule overlap or correlate. However, these metrics are, at best, proxies, and they depend strongly on various experimental choices. Here, we define a new strategy that leverages multispectral imaging and phasor analysis, termed the phasor mixing coefficient (PMC). The PMC measures the precise mixing of fluorescent signals in each pixel. We demonstrate how the PMC captures complex biological subtlety by offering two distinct values, a global measure of overall color mixing and the homogeneity thereof. We additionally show that the PMC exhibits less sensitivity to signal-to-noise ratio, intensity threshold and background signal compared to canonical methods. Moreover, this method provides a means to visualize color mixing at each pixel. We show that the PMC offers users a nuanced and robust metric to quantify biological association.

}, issn = {0021-9533}, doi = {10.1242/jcs.264388}, url = {https://doi.org/10.1242/jcs.264388}, author = {Puls, Owen F. and Aaron, Jesse S. and Quarles, Ellen K. and Khuon, Satya and Eisenman, Leanna R. and Kamaid, Andres and Malacrida, Leonel and Chew, Teng-Leong} } @article {69656, title = {Leukocytes use endothelial membrane tunnels to extravasate the vasculature}, journal = {Cell Rep}, year = {2025}, month = {2025 Sep 04}, abstract = {

Upon inflammation, leukocytes extravasate through endothelial cells. When they extravasate, it is generally accepted that neighboring endothelial cells disconnect. Careful examination of endothelial junctions showed a partial membrane overlap beyond VE-cadherin distribution. These overlaps are regulated by actin polymerization and, although marked by, do not require PECAM-1, nor VE-cadherin. Neutrophils prefer wider membrane overlaps as exit sites. Detailed 3D analysis of neutrophil transmigration in real time at high spatiotemporal resolution revealed that overlapping endothelial membranes form a tunnel during neutrophil transmigration. These tunnels are formed by the neutrophil lifting the membrane of the upper endothelial cell while indenting and crawling over the membrane of the underlying endothelial cell. Our work shows that endothelial cells do not simply retract upon the passage of neutrophils but provide membrane tunnels, allowing neutrophils to extravasate. This discovery defines the 3D multicellular architecture in which the paracellular transmigration of neutrophils occurs.

}, doi = {10.1016/j.celrep.2025.116242}, url = {https://doi.org/10.1016/j.celrep.2025.116242}, author = {van der Meer, Werner and van Steen, Abraham C.I. and Mahlandt, Eike and Rolas, Loic and Wang, Haitao and Arts, Janine and Kempers, Lanette and Gr{\"o}nloh, Max and Schoon, Rianne M. and Driessen, Amber and van Rijssel, Jos and Klaassen, Ingeborg and Schlingemann, Reinier O. and Manavski, Yosif and Hoogenboezem, Mark and Boon, Reinier A. and Khuon, Satya and Wait, Eric and Heddleston, John and Chew, Teng-Leong and Nolte, Martijn A and Nourshargh, Sussan and Goedhart, Joachim and van Buul, Jaap D} } @article {70198, title = {Redefining colocalization analysis with a novel Phasor Mixing Coefficient.}, journal = {J Cell Sci}, year = {2025}, month = {2025 Nov 24}, abstract = {

The first step to probing any potential interaction between two biomolecules is to determine their spatial association. In other words, if two biomolecules localize similarly within a cell, then it is plausible they could interact. Traditionally, this is quantified through various colocalization metrics. These measures infer this association by estimating the degree to which fluorescent signals from each biomolecule overlap or correlate. However, these metrics are, at best, proxies, and they depend strongly on various experimental choices. Alternatively, here we define a new strategy which leverages multispectral imaging and phasor analysis, termed the Phasor Mixing Coefficient (PMC). PMC measures the precise mixing of fluorescent signals in each pixel. We demonstrate how PMC captures complex biological subtlety by offering two distinct values, a global measure of overall color mixing and the homogeneity thereof. We additionally show that PMC exhibits less sensitivity to signal-to-noise ratio, intensity threshold, and background signal compared to canonical methods. Moreover, this method provides a means to visualize color mixing at each pixel. We show that PMC offers users a nuanced and robust metric to quantify biological association.

}, issn = {1477-9137}, doi = {10.1242/jcs.264388}, author = {Puls, Owen F and Aaron, Jesse S and Quarles, Ellen K and Khuon, Satya and Eisenman, Leanna R and Kamaid, Andres and Malacrida, Leonel and Chew, Teng-Leong} } @article {70203, title = {Redefining colocalization analysis with a novel Phasor Mixing Coefficient.}, journal = {J Cell Sci}, year = {2025}, month = {2025 Nov 24}, abstract = {

The first step to probing any potential interaction between two biomolecules is to determine their spatial association. In other words, if two biomolecules localize similarly within a cell, then it is plausible they could interact. Traditionally, this is quantified through various colocalization metrics. These measures infer this association by estimating the degree to which fluorescent signals from each biomolecule overlap or correlate. However, these metrics are, at best, proxies, and they depend strongly on various experimental choices. Alternatively, here we define a new strategy which leverages multispectral imaging and phasor analysis, termed the Phasor Mixing Coefficient (PMC). PMC measures the precise mixing of fluorescent signals in each pixel. We demonstrate how PMC captures complex biological subtlety by offering two distinct values, a global measure of overall color mixing and the homogeneity thereof. We additionally show that PMC exhibits less sensitivity to signal-to-noise ratio, intensity threshold, and background signal compared to canonical methods. Moreover, this method provides a means to visualize color mixing at each pixel. We show that PMC offers users a nuanced and robust metric to quantify biological association.

}, issn = {1477-9137}, doi = {10.1242/jcs.264388}, url = {https://doi.org/10.1242/jcs.264388}, author = {Puls, Owen F and Aaron, Jesse S and Quarles, Ellen K and Khuon, Satya and Eisenman, Leanna R and Kamaid, Andres and Malacrida, Leonel and Chew, Teng-Leong} } @article {69147, title = {Believing is seeing - the deceptive influence of bias in quantitative microscopy.}, journal = {Journal of Cell Science}, volume = {137}, year = {2024}, month = {2024 Jan 10}, abstract = {

The visual allure of microscopy makes it an intuitively powerful research tool. Intuition, however, can easily obscure or distort the reality of the information contained in an image. Common cognitive biases, combined with institutional pressures that reward positive research results, can quickly skew a microscopy project towards upholding, rather than rigorously challenging, a hypothesis. The impact of these biases on a variety of research topics is well known. What might be less appreciated are the many forms in which bias can permeate a microscopy experiment. Even well-intentioned researchers are susceptible to bias, which must therefore be actively recognized to be mitigated. Importantly, although image quantification has increasingly become an expectation, ostensibly to confront subtle biases, it is not a guarantee against bias and cannot alone shield an experiment from cognitive distortions. Here, we provide illustrative examples of the insidiously pervasive nature of bias in microscopy experiments - from initial experimental design to image acquisition, analysis and data interpretation. We then provide suggestions that can serve as guard rails against bias.

}, keywords = {Bias, Humans, Microscopy, Research Personnel}, issn = {1477-9137}, doi = {10.1242/jcs.261567}, author = {Lee, Rachel M and Eisenman, Leanna R and Khuon, Satya and Aaron, Jesse S and Chew, Teng-Leong} } @article {69442, title = {Coumarin as a general switching auxiliary to prepare photochromic and spontaneously blinking fluorophores}, journal = {bioRxiv}, year = {2024}, month = {2024 May 12}, abstract = {

Single-molecule localization microscopy (SMLM) uses activatable or switchable fluorophores to create non-diffraction limited maps of molecular location in biological samples. Despite the utility of this imaging technique, the portfolio of appropriate labels for SMLM remains limited. Here, we describe a general strategy for the construction of {\textquotedblleft}glitter bomb{\textquotedblright} labels by simply combining rhodamine and coumarin dyes though an amide bond. Condensation of the ortho-carboxyl group on the pendant phenyl ring of rhodamine dyes with a 7-aminocoumarin yields photochromic or spontaneously blinking fluorophores depending on the parent rhodamine structure. We apply this strategy to prepare labels useful super-resolution experiments in fixed cells using different attachment techniques. This general glitter bomb strategy should lead to improved labels for SMLM, ultimately enabling the creation of detailed molecular maps in biological samples.

}, doi = {10.1101/2024.05.12.593749}, url = {https://www.biorxiv.org/content/early/2024/05/12/2024.05.12.593749}, author = {Jradi, Fadi M. and English, Brian P. and Brown, Timothy A. and Aaron, Jesse and Khuon, Satya and Galbraith, James A. and Galbraith, Catherine G. and Lavis, Luke D.} } @article {69128, title = {Phase diversity-based wavefront sensing for fluorescence microscopy.}, journal = {Optica}, volume = {11}, year = {2024}, month = {2024 Jun 10}, pages = {806-820}, abstract = {

Fluorescence microscopy is an invaluable tool in biology, yet its performance is compromised when the wavefront of light is distorted due to optical imperfections or the refractile nature of the sample. Such optical aberrations can dramatically lower the information content of images by degrading image contrast, resolution, and signal. Adaptive optics (AO) methods can sense and subsequently cancel the aberrated wavefront, but are too complex, inefficient, slow, or expensive for routine adoption by most labs. Here we introduce a rapid, sensitive, and robust wavefront sensing scheme based on phase diversity, a method successfully deployed in astronomy but underused in microscopy. Our method enables accurate wavefront sensing to less than λ/35 root mean square (RMS) error with few measurements, and AO with no additional hardware besides a corrective element. After validating the method with simulations, we demonstrate calibration of a deformable mirror \> 100-fold faster than comparable methods (corresponding to wavefront sensing on the ~100 ms scale), and sensing and subsequent correction of severe aberrations (RMS wavefront distortion exceeding λ/2), restoring diffraction-limited imaging on extended biological samples.

}, doi = {10.1364/OPTICA.518559}, url = {https://doi.org/10.1364/OPTICA.518559}, author = {Johnson, Courtney and Guo, Min and Schneider, Magdalena C and Su, Yijun and Khuon, Satya and Reiser, Nikolaj and Wu, Yicong and Riviere, Patrick La and Shroff, Hari} } @article {68807, title = {Architecture and dynamics of a desmosome-endoplasmic reticulum complex.}, journal = {Nature Cell Biology}, volume = {25}, year = {2023}, month = {2023 Jun 16}, pages = {823-835}, abstract = {

The endoplasmic reticulum (ER) forms a dynamic network that contacts other cellular membranes to regulate stress responses, calcium signalling and lipid transfer. Here, using high-resolution volume electron microscopy, we find that the ER forms a previously unknown association with keratin intermediate filaments and desmosomal cell-cell junctions. Peripheral ER assembles into mirror image-like arrangements at desmosomes and exhibits nanometre proximity to keratin filaments and the desmosome cytoplasmic plaque. ER tubules exhibit stable associations with desmosomes, and perturbation of desmosomes or keratin filaments alters ER organization, mobility and expression of ER stress transcripts. These findings indicate that desmosomes and the keratin cytoskeleton regulate the distribution, function and dynamics of the ER network. Overall, this study reveals a previously unknown subcellular architecture defined by the structural integration of ER tubules with an epithelial intercellular junction.

}, keywords = {Cytoskeleton, Desmosomes, Endoplasmic Reticulum, Intermediate Filaments, Keratins}, issn = {1476-4679}, doi = {10.1038/s41556-023-01154-4}, author = {Bharathan, Navaneetha Krishnan and Giang, William and Hoffman, Coryn L and Aaron, Jesse S and Khuon, Satya and Chew, Teng-Leong and Preibisch, Stephan and Trautman, Eric T and Heinrich, Larissa and Bogovic, John and Bennett, Davis and Ackerman, David and Park, Woohyun and Petruncio, Alyson and Weigel, Aubrey V and Saalfeld, Stephan and Wayne Vogl, A and Stahley, Sara N and Kowalczyk, Andrew P} } @article {67989, title = {Quantifying Molecular Dynamics within Complex Cellular Morphologies using LLSM-FRAP.}, journal = {Small Methods}, year = {2022}, month = {2022 Jun 01}, pages = {e2200149}, abstract = {

Quantifying molecular dynamics within the context of complex cellular morphologies is essential toward understanding the inner workings and function of cells. Fluorescence recovery after photobleaching (FRAP) is one of the most broadly applied techniques to measure the reaction diffusion dynamics of molecules in living cells. FRAP measurements typically restrict themselves to single-plane image acquisition within a subcellular-sized region of interest due to the limited temporal resolution and undesirable photobleaching induced by 3D fluorescence confocal or widefield microscopy. Here, an experimental and computational pipeline combining lattice light sheet microscopy, FRAP, and numerical simulations, offering rapid and minimally invasive quantification of molecular dynamics with respect to 3D cell morphology is presented. Having the opportunity to accurately measure and interpret the dynamics of molecules in 3D with respect to cell morphology has the potential to reveal unprecedented insights into the function of living cells.

}, issn = {2366-9608}, doi = {10.1002/smtd.202200149}, author = {Colin-York, Huw and Heddleston, John and Wait, Eric and Karedla, Narain and DeSantis, Michael and Khuon, Satya and Chew, Teng-Leong and Sbalzarini, Ivo F and Fritzsche, Marco} } @article {67987, title = {When light meets biology - how the specimen affects quantitative microscopy.}, journal = {Journal of Cell Science}, volume = {135}, year = {2022}, month = {2022 Mar 15}, abstract = {

Fluorescence microscopy images should not be treated as perfect representations of biology. Many factors within the biospecimen itself can drastically affect quantitative microscopy data. Whereas some sample-specific considerations, such as photobleaching and autofluorescence, are more commonly discussed, a holistic discussion of sample-related issues (which includes less-routine topics such as quenching, scattering and biological anisotropy) is required to appropriately guide life scientists through the subtleties inherent to bioimaging. Here, we consider how the interplay between light and a sample can cause common experimental pitfalls and unanticipated errors when drawing biological conclusions. Although some of these discrepancies can be minimized or controlled for, others require more pragmatic considerations when interpreting image data. Ultimately, the power lies in the hands of the experimenter. The goal of this Review is therefore to survey how biological samples can skew quantification and interpretation of microscopy data. Furthermore, we offer a perspective on how to manage many of these potential pitfalls.

}, issn = {1477-9137}, doi = {10.1242/jcs.259656}, author = {Reiche, Michael A and Aaron, Jesse S and Boehm, Ulrike and DeSantis, Michael C and Hobson, Chad M and Khuon, Satya and Lee, Rachel M and Chew, Teng-Leong} } @article {67710, title = {Endothelial junctional membrane protrusions serve as hotspots for neutrophil transmigration.}, journal = {eLife}, volume = {10}, year = {2021}, month = {2021 Aug 25}, abstract = {

Upon inflammation, leukocytes rapidly transmigrate across the endothelium to enter the inflamed tissue. Evidence accumulates that leukocytes use preferred exit sites, though it is not yet clear how these hotspots in the endothelium are defined and how they are recognized by the leukocyte. Using lattice light sheet microscopy, we discovered that leukocytes prefer endothelial membrane protrusions at cell junctions for transmigration. Phenotypically, these junctional membrane protrusions are present in an asymmetric manner, meaning that one endothelial cell shows the protrusion and the adjacent one does not. Consequently, leukocytes cross the junction by migrating underneath the protruding endothelial cell. These protrusions depend on Rac1 activity and by using a photo-activatable Rac1 probe, we could artificially generate local exit-sites for leukocytes. Overall, we have discovered a new mechanism that uses local induced junctional membrane protrusions to facilitate/steer the leukocyte escape/exit from inflamed vessel walls.

}, issn = {2050-084X}, doi = {10.7554/eLife.66074}, author = {Arts, Janine Jg and Mahlandt, Eike K and Gr{\"o}nloh, Max and Schimmel, Lilian and Noordstra, Ivar and Gordon, Emma and van Steen, Abraham Ci and Tol, Simon and Walzog, Barbara and van Rijssel, Jos and Nolte, Martijn A and Postma, Marten and Khuon, Satya and Heddleston, John M and Wait, Eric and Chew, Teng Leong and Winter, Mark and Montanez, Eloi and Goedhart, Joachim and van Buul, Jaap D} } @article {67233, title = {ER-to-Golgi protein delivery through an interwoven, tubular network extending from ER.}, journal = {Cell}, volume = {184}, year = {2021}, month = {2021 Apr 29}, pages = {2412}, abstract = {

Cellular versatility depends on accurate trafficking of diverse proteins to their organellar destinations. For the secretory pathway (followed by approximately 30\% of all proteins), the physical nature of the vessel conducting the first portage (endoplasmic reticulum [ER] to Golgi apparatus) is unclear. We provide a dynamic 3D view of early secretory compartments in mammalian cells with isotropic resolution and precise protein localization using whole-cell, focused ion beam scanning electron microscopy with cryo-structured illumination microscopy and live-cell synchronized cargo release approaches. Rather than vesicles alone, the ER spawns an elaborate, interwoven tubular network of contiguous lipid bilayers (ER exit site) for protein export. This receptacle is capable of extending microns along microtubules while still connected to the ER by a thin neck. COPII localizes to this neck region and dynamically regulates cargo entry from the ER, while COPI acts more distally, escorting the detached, accelerating tubular entity on its way to joining the Golgi apparatus through microtubule-directed movement.

}, issn = {1097-4172}, doi = {10.1016/j.cell.2021.03.035}, author = {Weigel, Aubrey V and Chang, Chi-Lun and Shtengel, Gleb and Xu, C Shan and Hoffman, David P and Freeman, Melanie and Iyer, Nirmala and Aaron, Jesse and Khuon, Satya and Bogovic, John and Qiu, Wei and Hess, Harald F and Lippincott-Schwartz, Jennifer} } @article {67670, title = {Imaging Africa: a strategic approach to optical microscopy training in Africa.}, journal = {Nature Methods}, volume = {18}, year = {2021}, month = {2021 Aug 20}, pages = {847-855}, issn = {1548-7105}, doi = {10.1038/s41592-021-01227-y}, author = {Reiche, Michael A and Warner, Digby F and Aaron, Jesse and Khuon, Satya and Fletcher, Daniel A and Hahn, Klaus and Rogers, Kelly L and Mhlanga, Musa and Koch, Anastasia and Quaye, Wendye and Chew, Teng-Leong} } @article {64852, title = {Cytoskeletal control of antigen-dependent T cell activation.}, journal = {Cell Reports}, volume = {26}, year = {2019}, month = {2019 Mar 19}, pages = {3369-3379.e5}, abstract = {

Cytoskeletal actin dynamics is essential for T\ cell activation. Here, we show evidence that the binding kinetics of the antigen engaging the T\ cell receptor influences the nanoscale actin organization and mechanics of the immune synapse. Using an engineered T\ cell system expressing a specific T\ cell receptor and stimulated by a range of antigens, we found that the peak force experienced by the T\ cell receptor during activation was independent of the unbinding kinetics of the stimulating antigen. Conversely, quantification of the actin retrograde flow velocity at the synapse revealed a striking dependence on the antigen unbinding kinetics. These findings suggest that the dynamics of the actin cytoskeleton actively adjusted to normalize the force experienced by the T\ cell receptor in an antigen-specific manner. Consequently, tuning actin dynamics in response to antigen kinetics may thus\ be a mechanism that allows T\ cells to adjust the lengthscale and timescale of T\ cell receptor signaling.

}, issn = {2211-1247}, doi = {10.1016/j.celrep.2019.02.074}, author = {Colin-York, Huw and Javanmardi, Yousef and Skamrahl, Mark and Kumari, Sudha and Chang, Veronica T and Khuon, Satya and Taylor, Aaron and Chew, Teng-Leong and Betzig, Eric and Moeendarbary, Emad and Cerundolo, Vincenzo and Eggeling, Christian and Fritzsche, Marco} } @article {49467, title = {Development of 2-colour and 3D SMLM data analysis methods for fibrous spatial point patterns.}, journal = {Journal of Physics D: Applied Physics}, volume = {52}, year = {2018}, month = {2018 Oct 11}, pages = {1}, abstract = {

Abstract ingle molecule localisation microscopy (SMLM), experimentally achieved over a decade ago, has become a routinely used analytical tool across the life sciences. Synergistic advances in probe chemistry, optical physics and data analysis has propelled SMLM into the quantitative realm, enabling unprecedented access to the cellular machinery at the nanoscale. In its early years, SMLM primarily served as a platform for impressive rendered images of sub diffraction scale structures, however more recently a shift towards interrogating SMLM point pattern data in a robust mathematical framework has occurred. A prevalent theme in the SMLM field is the need for quantitative analytical methods, to better understand the underlying processes on which SMLM reports and to extract statistically valid biological insights. Whilst some forms of post processing analytics, for example cluster analysis, have been widely studied, others such as fibre analysis remain in their infancy. Here, we review the current state of the art of cluster analysis and fibre analysis and present new methods for their implementation in both 3D SMLM data sets and multi-colour data.

}, doi = {10.1088/1361-6463/aae7ac}, author = {Peters, Ruby and Griffi{\'e}, Juliette and Williamson, David and Aaron, Jesse and Khuon, Satya and Owen, Dylan} } @article {48621, title = {3D Bayesian cluster analysis of super-resolution data reveals LAT recruitment to the T cell synapse.}, journal = {Scientific Reports}, volume = {7}, year = {2017}, month = {2017 Jun 22}, pages = {4077}, abstract = {

Single-molecule localisation microscopy (SMLM) allows the localisation of fluorophores with a precision of 10-30 nm, revealing the cell{\textquoteright}s nanoscale architecture at the molecular level. Recently, SMLM has been extended to 3D, providing a unique insight into cellular machinery. Although cluster analysis techniques have been developed for 2D SMLM data sets, few have been applied to 3D. This lack of quantification tools can be explained by the relative novelty of imaging techniques such as interferometric photo-activated localisation microscopy (iPALM). Also, existing methods that could be extended to 3D SMLM are usually subject to user defined analysis parameters, which remains a major drawback. Here, we present a new open source cluster analysis method for 3D SMLM data, free of user definable parameters, relying on a model-based Bayesian approach which takes full account of the individual localisation precisions in all three dimensions. The accuracy and reliability of the method is validated using simulated data sets. This tool is then deployed on novel experimental data as a proof of concept, illustrating the recruitment of LAT to the T-cell immunological synapse in data acquired by iPALM providing ~10 nm isotropic resolution.

}, issn = {2045-2322}, doi = {10.1038/s41598-017-04450-w}, author = {Griffi{\'e}, Juliette and Shlomovich, Leigh and Williamson, David J and Shannon, Michael and Aaron, Jesse and Khuon, Satya and L Burn, Garth and Boelen, Lies and Peters, Ruby and Cope, Andrew P and Cohen, Edward A K and Rubin-Delanchy, Patrick and Owen, Dylan M} } @article {47709, title = {Super-resolution spectroscopic microscopy via photon localization.}, journal = {Nature Communications}, volume = {7}, year = {2016}, month = {2016 Jul 25}, pages = {12290}, abstract = {

Traditional photon localization microscopy analyses only the spatial distributions of photons emitted by individual molecules to reconstruct super-resolution optical images. Unfortunately, however, the highly valuable spectroscopic information from these photons have been overlooked. Here we report a spectroscopic photon localization microscopy that is capable of capturing the inherent spectroscopic signatures of photons from individual stochastic radiation events. Spectroscopic photon localization microscopy achieved higher spatial resolution than traditional photon localization microscopy through spectral discrimination to identify the photons emitted from individual molecules. As a result, we resolved two fluorescent molecules, which were 15 nm apart, with the corresponding spatial resolution of 10 nm-a four-fold improvement over photon localization microscopy. Using spectroscopic photon localization microscopy, we further demonstrated simultaneous multi-colour super-resolution imaging of microtubules and mitochondria in COS-7 cells and showed that background autofluorescence can be identified through its distinct emission spectra.

}, issn = {2041-1723}, doi = {10.1038/ncomms12290}, author = {Dong, Biqin and Almassalha, Luay and Urban, Ben E and Nguyen, The-Quyen and Khuon, Satya and Chew, Teng-Leong and Backman, Vadim and Sun, Cheng and Zhang, Hao F} }