@article {70185, title = {The CENP-A chaperone complex spatially organizes centromeres}, journal = {bioRxiv}, year = {2025}, month = {2026 Nov 09}, abstract = {

Centromeres, defined by CENP-A-containing nucleosomes, direct the assembly of kinetochores for spindle attachment. In mitosis, CENP-A and the constitutive centromere-associated network (CCAN) of the inner kinetochore are arranged into bipartite subdomains within clearings of chromatin. However, it remains unclear whether any of these features exist before mitosis. We show that in interphase, CENP-A and the CCAN assemble \~200-300 nm shell-like structures that enclose a chromatin-poor central cavity. Strikingly, this cavity is occupied by the interphase-specific CENP-A chaperone complex, which promotes CENP-A assembly once per cell cycle. However, chaperone presence, but not CENP-A incorporation, is required to generate both the shell architecture and the chromatin clearing. The CCAN scaffold CENP-C, which links CENP-A nucleosomes to the chaperone complex, exhibits radial organization spanning the entire structure and is essential for its formation. These data uncover a previously unrecognized structural role for the CENP-A chaperone machinery in establishing interphase centromere architecture and suggest a mechanism by which this machinery configures centromeres for faithful kinetochore assembly and genome stability.

}, doi = {10.1101/2025.11.07.687291}, url = {https://www.biorxiv.org/content/early/2025/11/09/2025.11.07.687291}, author = {Gupta, Hindol and Haase, Julian and Wu, Yicong and Chen, Jiji and Karadimov, George and Shroff, Hari and Kelly, Alexander E.} } @article {69719, title = {Deep learning-based aberration compensation improves contrast and resolution in fluorescence microscopyAbstract}, journal = {Nature Communications}, volume = {16}, year = {2025}, month = {Jan-12-2025}, abstract = {

Optical aberrations hinder fluorescence microscopy of thick samples, reducing image signal, contrast, and resolution. Here we introduce a deep learning-based strategy for aberration compensation, improving image quality without slowing image acquisition, applying additional dose, or introducing more optics. Our method (i) introduces synthetic aberrations to images acquired on the shallow side of image stacks, making them resemble those acquired deeper into the volume and (ii) trains neural networks to reverse the effect of these aberrations. We use simulations and experiments to show that applying the trained {\textquoteleft}de-aberration{\textquoteright} networks outperforms alternative methods, providing restoration on par with adaptive optics techniques; and subsequently apply the networks to diverse datasets captured with confocal, light-sheet, multi-photon, and super-resolution microscopy. In all cases, the improved quality of the restored data facilitates qualitative image inspection and improves downstream image quantitation, including orientational analysis of blood vessels in mouse tissue and improved membrane and nuclear segmentation in\ C. elegans\ embryos.

}, doi = {10.1038/s41467-024-55267-x}, url = {https://www.nature.com/articles/s41467-024-55267-xhttps://www.nature.com/articles/s41467-024-55267-x.pdfhttps://www.nature.com/articles/s41467-024-55267-x.pdfhttps://www.nature.com/articles/s41467-024-55267-x}, author = {Guo, Min and Wu, Yicong and Hobson, Chad M. and Su, Yijun and Qian, Shuhao and Krueger, Eric and Christensen, Ryan and Kroeschell, Grant and Bui, Johnny and Chaw, Matthew and Zhang, Lixia and Liu, Jiamin and Hou, Xuekai and Han, Xiaofei and Lu, Zhiye and Ma, Xuefei and Zhovmer, Alexander and Combs, Christian and Moyle, Mark and Yemini, Eviatar and Liu, Huafeng and Liu, Zhiyi and Benedetto, Alexandre and La Riviere, Patrick and Col{\'o}n-Ramos, Daniel and Shroff, Hari} } @article {69720, title = {HD2Net: A deep learning framework for simultaneous denoising and deaberration in fluorescence microscopy}, journal = {bioRxiv}, year = {2025}, month = {01/2025}, abstract = {

Fluorescence microscopy is essential for biological research, offering high-contrast imaging of microscopic structures. However, the quality of these images is often compromised by optical aberrations and noise, particularly in low signal-to-noise ratio (SNR) conditions. While adaptive optics (AO) can correct aberrations, it requires costly hardware and slows down imaging; whereas current denoising approaches boost the SNR but leave out the aberration compensation. To address these limitations, we introduce HD2Net, a deep learning framework that enhances image quality by simultaneously denoising and suppressing the effect of aberrations without the need for additional hardware. Building on our previous work, HD2Net incorporates noise estimation and aberration removal modules, effectively restoring images degraded by noise and aberrations. Through comprehensive evaluation of synthetic phantoms and biological data, we demonstrate that HD2Net outperforms existing methods, significantly improving image resolution and contrast. This framework offers a promising solution for enhancing biological imaging, particularly in challenging aberrating and low-light conditions.

}, doi = {10.1101/2025.01.06.631475}, url = {https://www.biorxiv.org/content/early/2025/01/08/2025.01.06.631475}, author = {Hou, Xuekai and Li, Yue and Hobson, Chad M. and Shroff, Hari and Guo, Min and Liu, Huafeng} } @article {69734, title = {HD2Net: A Deep Learning Framework for Simultaneous Denoising and Deaberration in Fluorescence Microscopy}, journal = {bioRxiv}, year = {2025}, month = {2025 Jan 8}, abstract = {

Fluorescence microscopy is essential for biological research, offering high-contrast imaging of microscopic structures. However, the quality of these images is often compromised by optical aberrations and noise, particularly in low signal-to-noise ratio (SNR) conditions. While adaptive optics (AO) can correct aberrations, it requires costly hardware and slows down imaging; whereas current denoising approaches boost the SNR but leave out the aberration compensation. To address these limitations, we introduce HD2Net, a deep learning framework that enhances image quality by simultaneously denoising and suppressing the effect of aberrations without the need for additional hardware. Building on our previous work, HD2Net incorporates noise estimation and aberration removal modules, effectively restoring images degraded by noise and aberrations. Through comprehensive evaluation of synthetic phantoms and biological data, we demonstrate that HD2Net outperforms existing methods, significantly improving image resolution and contrast. This framework offers a promising solution for enhancing biological imaging, particularly in challenging aberrating and low-light conditions.

}, doi = {10.1101/2025.01.06.631475}, url = {https://www.biorxiv.org/content/early/2025/01/08/2025.01.06.631475}, author = {Hou, Xuekai and Li, Yue and Hobson, Chad M. and Shroff, Hari and Guo, Min and Liu, Huafeng} } @article {69773, title = {Live imaging of the extracellular matrix with a glycan-binding fluorophore.}, journal = {Nat Methods}, year = {2025}, month = {2025 Feb 06}, abstract = {

All multicellular systems produce and dynamically regulate extracellular matrices (ECMs) that play essential roles in both biochemical and mechanical signaling. Though the spatial arrangement of these extracellular assemblies is critical to their biological functions, visualization of ECM structure is challenging, in part because the biomolecules that compose the ECM are difficult to fluorescently label individually and collectively. Here, we present a cell-impermeable small-molecule fluorophore, termed Rhobo6, that turns on and red shifts upon reversible binding to glycans. Given that most ECM components are densely glycosylated, the dye enables wash-free visualization of ECM, in systems ranging from in vitro substrates to in vivo mouse mammary tumors. Relative to existing techniques, Rhobo6 provides a broad substrate profile, superior tissue penetration, non-perturbative labeling, and negligible photobleaching. This work establishes a straightforward method for imaging the distribution of ECM in live tissues and organisms, lowering barriers for investigation of extracellular biology.

}, issn = {1548-7105}, doi = {10.1038/s41592-024-02590-2}, url = {https://doi.org/10.1038/s41592-024-02590-2}, author = {Fiore, Antonio and Yu, Guoqiang and Northey, Jason J and Patel, Ronak and Ravenscroft, Thomas A. and Ikegami, Richard and Kolkman, W and Pratik Kumar and Dilan, Tanya L and Ruetten, Virginia M.S. and Ahrens, Misha B and Shroff, Hari and Wang, Shaohe and Weaver, Valarie M and Pedram, Kayvon} } @article {69959, title = {Resolution in super-resolution microscopy - facts, artifacts, technological advancements and biological applications}, journal = {J Cell Sci}, volume = {138}, year = {2025}, month = {2025 May 27}, abstract = {

Super-resolution microscopy (SRM) has undeniable potential for scientific discovery, yet still presents many challenges that hinder its widespread adoption, including technical trade-offs between resolution, speed and photodamage, as well as limitations in imaging live samples and larger, more complex biological structures. Furthermore, SRM often requires specialized expertise and complex instrumentation, which can deter biologists from fully embracing the technology. In this Perspective, a follow-up to our recent Q\&A article, we aim to demystify these challenges by addressing common questions and misconceptions surrounding SRM. Experts offer practical insights into how biologists can maximize the benefits of SRM while navigating issues such as photobleaching, image artifacts and the limitations of existing techniques. We also highlight recent developments in SRM that continue to push the boundaries of resolution. Our goal is to equip researchers with the crucial knowledge they need to harness the full potential of SRM.

}, keywords = {Animals, Artifacts, Humans, Microscopy, Microscopy, Fluorescence}, issn = {1477-9137}, doi = {10.1242/jcs.263567}, url = {https://doi.org/10.1242/jcs.263567}, author = {Prakash, Kirti and Baddeley, David and Eggeling, Christian and Fiolka, Reto and Heintzmann, Rainer and Manley, Suliana and Radenovic, Aleksandra and Shroff, Hari and Smith, Carlas and Schermelleh, Lothar} } @article {69792, title = {Volumetric imaging of the 3D orientation of cellular structures with a polarized fluorescence light-sheet microscope.}, journal = {Proc Natl Acad Sci }, volume = {122}, year = {2025}, month = {2025 Feb 25}, pages = {e2406679122}, abstract = {

Polarized fluorescence microscopy is a valuable tool for measuring molecular orientations in biological samples, but techniques for recovering three-dimensional orientations and positions of fluorescent ensembles are limited. We report a polarized dual-view light-sheet system for determining the diffraction-limited three-dimensional distribution of the orientations and positions of ensembles of fluorescent dipoles that label biological structures. We share a set of visualization, histogram, and profiling tools for interpreting these positions and orientations. We model the distributions based on the polarization-dependent efficiency of excitation and detection of emitted fluorescence, using coarse-grained representations we call orientation distribution functions (ODFs). We apply ODFs to create physics-informed models of image formation with spatio-angular point-spread and transfer functions. We use theory and experiment to conclude that light-sheet tilting is a necessary part of our design for recovering all three-dimensional orientations. We use our system to extend known two-dimensional results to three dimensions in FM1-43-labeled giant unilamellar vesicles, fast-scarlet-labeled cellulose in xylem cells, and phalloidin-labeled actin in U2OS cells. Additionally, we observe phalloidin-labeled actin in mouse fibroblasts grown on grids of labeled nanowires and identify correlations between local actin alignment and global cell-scale orientation, indicating cellular coordination across length scales.

}, keywords = {Actins, Animals, Humans, Imaging, Three-Dimensional, Mice, Microscopy, Fluorescence, Microscopy, Polarization, Xylem}, issn = {1091-6490}, doi = {10.1073/pnas.2406679122}, author = {Chandler, Talon and Guo, Min and Su, Yijun and Chen, Jiji and Wu, Yicong and Liu, Junyu and Agashe, Atharva and Fischer, Robert S and Mehta, Shalin B and Kumar, Abhishek and Baskin, Tobias I and Jaumouill{\'e}, Valentin and Liu, Huafeng and Swaminathan, Vinay and Nain, Amrinder S and Oldenbourg, Rudolf and La Riviere, Patrick J and Shroff, Hari} } @article {69297, title = {Cell division machinery drives cell-specific gene activation during differentiation in .}, journal = {Proc Natl Acad Sci U S A}, volume = {121}, year = {2024}, month = {2024 Mar 6}, pages = {e2400584121}, abstract = {

When faced with starvation, the bacterium transforms itself into a dormant cell type called a "spore". Sporulation initiates with an asymmetric division event, which requires the relocation of the core divisome components FtsA and FtsZ, after which the sigma factor σ is exclusively activated in the smaller daughter cell. Compartment-specific activation of σ requires the SpoIIE phosphatase, which displays a biased localization on one side of the asymmetric division septum and associates with the structural protein DivIVA, but the mechanism by which this preferential localization is achieved is unclear. Here, we isolated a variant of DivIVA that indiscriminately activates σ in both daughter cells due to promiscuous localization of SpoIIE, which was corrected by overproduction of FtsA and FtsZ. We propose that the core components of the redeployed cell division machinery drive the asymmetric localization of DivIVA and SpoIIE to trigger the initiation of the sporulation program.

}, issn = {1091-6490}, doi = {10.1073/pnas.2400584121}, author = {Chareyre, Sylvia and Li, Xuesong and Anjuwon-Foster, Brandon R and Updegrove, Taylor B and Clifford, Sarah and Brogan, Anna P and Su, Yijun and Zhang, Lixia and Chen, Jiji and Shroff, Hari and Ramamurthi, Kumaran S} } @article {69558, title = {Convolutional neural network transformer (CNNT) for fluorescence microscopy image denoising with improved generalization and fast adaptation.}, journal = {Sci Rep}, volume = {14}, year = {2024}, month = {2024 Aug 06}, pages = {18184}, abstract = {

Deep neural networks can improve the quality of fluorescence microscopy images. Previous methods, based on Convolutional Neural Networks (CNNs), require time-consuming training of individual models for each experiment, impairing their applicability and generalization. In this study, we propose a novel imaging-transformer based model, Convolutional Neural Network Transformer (CNNT), that outperforms CNN based networks for image denoising. We train a general CNNT based backbone model from pairwise high-low Signal-to-Noise Ratio (SNR) image volumes, gathered from a single type of fluorescence microscope, an instant Structured Illumination Microscope. Fast adaptation to new microscopes is achieved by fine-tuning the backbone on only 5-10 image volume pairs per new experiment. Results show that the CNNT backbone and fine-tuning scheme significantly reduces training time and improves image quality, outperforming models trained using only CNNs such as 3D-RCAN and Noise2Fast. We show three examples of efficacy of this approach in wide-field, two-photon, and confocal fluorescence microscopy.

}, issn = {2045-2322}, doi = {10.1038/s41598-024-68918-2}, url = {https://doi.org/10.1038/s41598-024-68918-2}, author = {Rehman, Azaan and Zhovmer, Alexander and Sato, Ryo and Mukouyama, Yoh-Suke and Chen, Jiji and Rissone, Alberto and Puertollano, Rosa and Liu, Jiamin and Vishwasrao, Harshad D and Shroff, Hari and Combs, Christian A and Xue, Hui} } @article {69440, title = {Imaging the extracellular matrix in live tissues and organisms with a glycan-binding fluorophore}, journal = {bioRxiv}, year = {2024}, month = {2024 May 10}, abstract = {

All multicellular systems produce and dynamically regulate extracellular matrices (ECM) that play important roles in both biochemical and mechanical signaling. Though the spatial arrangement of these extracellular assemblies is critical to their biological functions, visualization of ECM structure is challenging, in part because the biomolecules that compose the ECM are difficult to fluorescently label individually and collectively. Here, we present a cell-impermeable small molecule fluorophore, termed Rhobo6, that turns on and red shifts upon reversible binding to glycans. Given that most ECM components are densely glycosylated, the dye enables wash-free visualization of ECM, in systems ranging from in vitro substrates to in vivo mouse mammary tumors. Relative to existing techniques, Rhobo6 provides a broad substrate profile, superior tissue penetration, nonperturbative labeling, and negligible photobleaching. This work establishes a straightforward method for imaging the distribution of ECM in live tissues and organisms, lowering barriers for investigation of extracellular biology.

}, doi = {10.1101/2024.05.09.593460}, url = {https://www.biorxiv.org/content/early/2024/05/10/2024.05.09.593460}, author = {Fiore, Antonio and Yu, Guoqiang and Northey, Jason J. and Patel, Ronak and Ravenscroft, Thomas A. and Ikegami, Richard and Kolkman, Wiert and Kumar, Pratik and Grimm, Jonathan B. and Dilan, Tanya L. and Ruetten, Virginia M.S. and Ahrens, Misha B. and Shroff, Hari and Lavis, Luke D. and Wang, Shaohe and Weaver, Valerie M. and Pedram, Kayvon} } @article {69226, title = {Live-cell imaging powered by computation.}, journal = {Nature Reviews Molecular Cell Biology}, year = {2024}, month = {2024 Feb 20}, abstract = {

The proliferation of microscopy methods for live-cell imaging offers many new possibilities for users but can also be challenging to navigate. The prevailing challenge in live-cell fluorescence microscopy is capturing intra-cellular dynamics while preserving cell viability. Computational methods can help to address this challenge and are now shifting the boundaries of what is possible to capture in living systems. In this Review, we discuss these computational methods focusing on artificial intelligence-based approaches that can be layered on top of commonly used existing microscopies as well as hybrid methods that integrate computation and microscope hardware. We specifically discuss how computational approaches can improve the signal-to-noise ratio, spatial resolution, temporal resolution and multi-colour capacity of live-cell imaging.

}, issn = {1471-0080}, doi = {10.1038/s41580-024-00702-6}, author = {Shroff, Hari and Testa, Ilaria and Jug, Florian and Manley, Suliana} } @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 {69517, title = {Resolution in super-resolution microscopy - definition, trade-offs and perspectives.}, journal = {Nat Rev Mol Cell Biol}, year = {2024}, month = {2024 Jul 01}, abstract = {

Super-resolution microscopy (SRM) is gaining popularity in biosciences; however, claims about optical resolution are contested and often misleading. In this Viewpoint, experts share their views on resolution and common trade-offs, such as labelling and post-processing, aiming to clarify them for biologists and facilitate deeper understanding and best use of SRM.

}, issn = {1471-0080}, doi = {10.1038/s41580-024-00755-7}, author = {Prakash, Kirti and Baddeley, David and Eggeling, Christian and Fiolka, Reto and Heintzmann, Rainer and Manley, Suliana and Radenovic, Aleksandra and Smith, Carlas and Shroff, Hari and Schermelleh, Lothar} } @article {68409, title = {Practical considerations for quantitative light sheet fluorescence microscopy.}, journal = {Nature Methods}, volume = {19}, year = {2022}, month = {2022 Dec 01}, pages = {1538-49}, abstract = {

Fluorescence microscopy has evolved from a purely observational tool to a platform for quantitative, hypothesis-driven research. As such, the demand for faster and less phototoxic imaging modalities has spurred a rapid growth in light sheet fluorescence microscopy (LSFM). By restricting the excitation to a thin plane, LSFM reduces the overall light dose to a specimen while simultaneously improving image contrast. However, the defining characteristics of light sheet microscopes subsequently warrant unique considerations in their use for quantitative experiments. In this Perspective, we outline many of the pitfalls in LSFM that can compromise analysis and confound interpretation. Moreover, we offer guidance in addressing these caveats when possible. In doing so, we hope to provide a useful resource for life scientists seeking to adopt LSFM to quantitatively address complex biological hypotheses.

}, issn = {1548-7105}, doi = {10.1038/s41592-022-01632-x}, author = {Hobson, Chad M and Guo, Min and Vishwasrao, Harshad D and Wu, Yicong and Shroff, Hari and Chew, Teng-Leong} } @article {48595, title = {mRNA quantification using single-molecule FISH in Drosophila embryos.}, journal = {Nature Protocols}, volume = {12}, year = {2017}, month = {2017 Jul}, pages = {1326-1348}, abstract = {

Spatial information is critical to the interrogation of developmental and tissue-level regulation of gene expression. However, this information is usually lost when global mRNA levels from tissues are measured using reverse transcriptase PCR, microarray analysis or high-throughput sequencing. By contrast, single-molecule fluorescence in situ hybridization (smFISH) preserves the spatial information of the cellular mRNA content with subcellular resolution within tissues. Here we describe an smFISH protocol that allows for the quantification of single mRNAs in Drosophila embryos, using commercially available smFISH probes (e.g., short fluorescently labeled DNA oligonucleotides) in combination with wide-field epifluorescence, confocal or instant structured illumination microscopy (iSIM, a super-resolution imaging approach) and a spot-detection algorithm. Fixed Drosophila embryos are hybridized in solution with a mixture of smFISH probes, mounted onto coverslips and imaged in 3D. Individual fluorescently labeled mRNAs are then localized within tissues and counted using spot-detection software to generate quantitative, spatially resolved gene expression data sets. With minimum guidance, a graduate student can successfully implement this protocol. The smFISH procedure described here can be completed in 4-5 d.

}, issn = {1750-2799}, doi = {10.1038/nprot.2017.030}, author = {Trcek, Tatjana and Lionnet, Timoth{\'e}e and Shroff, Hari and Lehmann, Ruth} } @article {48731, title = {Myc Regulates Chromatin Decompaction and Nuclear Architecture during B Cell Activation.}, journal = {Molecular Cell}, volume = {67}, year = {2017}, month = {2017 Jul 28}, pages = {566-78}, abstract = {

50 years ago, Vincent Allfrey and colleagues discovered that lymphocyte activation triggers massive acetylation of chromatin. However, the molecular mechanisms driving epigenetic accessibility are still unknown. We here show that stimulated lymphocytes decondense chromatin by three differentially regulated steps. First, chromatin is repositioned away from the nuclear periphery in response to global acetylation. Second, histone nanodomain clusters decompact into mononucleosome fibers through a mechanism that requires Myc and continual energy input. Single-molecule imaging shows that this step lowers transcription factor residence time and non-specific collisions during sampling for DNA targets. Third, chromatin interactions shift from long range to predominantly short range, and CTCF-mediated loops and contact domains double in numbers. This architectural change facilitates cognate promoter-enhancer contacts and also requires Myc and continual ATP production. Our results thus define the nature and transcriptional impact of chromatin decondensation and reveal an unexpected role for Myc in the establishment of nuclear topology in mammalian cells.

}, issn = {1097-4164}, doi = {10.1016/j.molcel.2017.07.013}, author = {Kieffer-Kwon, Kyong-Rim and Nimura, Keisuke and Rao, Suhas S P and Xu, Jianliang and Jung, Seolkyoung and Pekowska, Aleksandra and Dose, Marei and Stevens, Evan and Mathe, Ewy and Dong, Peng and Huang, Su-Chen and Ricci, Maria Aurelia and Baranello, Laura and Zheng, Ying and Ardori, Francesco Tomassoni and Resch, Wolfgang and Stavreva, Diana and Nelson, Steevenson and McAndrew, Michael and Casellas, Adriel and Finn, Elizabeth and Gregory, Charles and St Hilaire, Brian Glenn and Johnson, Steven M and Dubois, Wendy and Cosma, Maria Pia and Batchelor, Eric and Levens, David and Phair, Robert D and Misteli, Tom and Tessarollo, Lino and Hager, Gordon and Lakadamyali, Melike and Liu, Zhe and Floer, Monique and Shroff, Hari and Aiden, Erez Lieberman and Casellas, Rafael} } @article {46975, title = {Drosophila germ granules are structured and contain homotypic mRNA clusters.}, journal = {Nature Communications}, volume = {6}, year = {2015}, month = {2015 Aug 5}, pages = {7962}, abstract = {

Germ granules, specialized ribonucleoprotein particles, are a hallmark of all germ cells. In Drosophila, an estimated 200 mRNAs are enriched in the germ plasm, and some of these have important, often conserved roles in germ cell formation, specification, survival and migration. How mRNAs are spatially distributed within a germ granule and whether their position defines functional properties is unclear. Here we show, using single-molecule FISH and structured illumination microscopy, a super-resolution approach, that mRNAs are spatially organized within the granule whereas core germ plasm proteins are distributed evenly throughout the granule. Multiple copies of single mRNAs organize into {\textquoteright}homotypic clusters{\textquoteright} that occupy defined positions within the center or periphery of the granule. This organization, which is maintained during embryogenesis and independent of the translational or degradation activity of mRNAs, reveals new regulatory mechanisms for germ plasm mRNAs that may be applicable to other mRNA granules.

}, issn = {2041-1723}, doi = {10.1038/ncomms8962}, author = {Trcek, Tatjana and Grosch, Markus and York, Andrew and Shroff, Hari and Lionnet, Timoth{\'e}e and Lehmann, Ruth} } @article {2046, title = {Single-molecule discrimination of discrete perisynaptic and distributed sites of actin filament assembly within dendritic spines. (With commentary)}, journal = {Neuron}, volume = {67}, year = {2010}, month = {2010 Jul 15}, pages = {86-99}, abstract = {

Within dendritic spines, actin is presumed to anchor receptors in the postsynaptic density and play numerous roles regulating synaptic transmission. However, the submicron dimensions of spines have hindered examination of actin dynamics within them and prevented live-cell discrimination of perisynaptic actin filaments. Using photoactivated localization microscopy, we measured movement of individual actin molecules within living spines. Velocity of single actin molecules along filaments, an index of filament polymerization rate, was highly heterogeneous within individual spines. Most strikingly, molecular velocity was elevated in discrete, well-separated foci occurring not principally at the spine tip, but in subdomains throughout the spine, including the neck. Whereas actin velocity on filaments at the synapse was substantially elevated, at the endocytic zone there was no enhanced polymerization activity. We conclude that actin subserves spatially diverse, independently regulated processes throughout spines. Perisynaptic actin forms a uniquely dynamic structure well suited for direct, active regulation of the synapse.

Commentary: A nice application of single particle tracking PALM (sptPALM), showing the flow of actin in the spines of live cultured neurons. Since 2008, the PALM in our lab has largely become a user facility, available to outside users as well as Janelians. Grad student Nick Frost in Tom Blanpied{\textquoteright}s group at the U. of Maryland Med School visited on a number of occasions to use the PALM, with training and assistance from Hari.

}, keywords = {Animals, Biological, Cells, Cultured, Dendritic Spines, Embryo, Green Fluorescent Proteins, Hippocampus, Mammalian, Microfilaments, Models, Nerve Tissue Proteins, Neuronal Plasticity, Neurons, Presynaptic Terminals, Protein Transport, Rats, Sprague-Dawley, Synaptic Transmission, Tissue Distribution, Transfection}, issn = {1097-4199}, doi = {10.1016/j.neuron.2010.05.026}, url = {http://www.ncbi.nlm.nih.gov/pubmed/20624594}, author = {Frost, Nicholas A and Shroff, Hari and Kong, Huihui and Betzig, Eric and Blanpied, Thomas A} } @article {2076, title = {Self-organization of the Escherichia coli chemotaxis network imaged with super-resolution light microscopy. (With commentary)}, journal = {PLoS Biology}, volume = {7}, year = {2009}, month = {2009 Jun 16}, pages = {e1000137}, abstract = {

The Escherichia coli chemotaxis network is a model system for biological signal processing. In E. coli, transmembrane receptors responsible for signal transduction assemble into large clusters containing several thousand proteins. These sensory clusters have been observed at cell poles and future division sites. Despite extensive study, it remains unclear how chemotaxis clusters form, what controls cluster size and density, and how the cellular location of clusters is robustly maintained in growing and dividing cells. Here, we use photoactivated localization microscopy (PALM) to map the cellular locations of three proteins central to bacterial chemotaxis (the Tar receptor, CheY, and CheW) with a precision of 15 nm. We find that cluster sizes are approximately exponentially distributed, with no characteristic cluster size. One-third of Tar receptors are part of smaller lateral clusters and not of the large polar clusters. Analysis of the relative cellular locations of 1.1 million individual proteins (from 326 cells) suggests that clusters form via stochastic self-assembly. The super-resolution PALM maps of E. coli receptors support the notion that stochastic self-assembly can create and maintain approximately periodic structures in biological membranes, without direct cytoskeletal involvement or active transport.

Commentary: Our goal as tool developers is to invent methods capable of uncovering new biological insights unobtainable by pre-existing technologies. A terrific example is given by this paper, where grad students Derek Greenfield and Ann McEvoy in Jan Liphardt{\textquoteright}s group at Berkeley used our PALM to image the size and position distributions of chemotaxis proteins in E. Coli with unprecedented precision and sensitivity. Their analysis revealed that the cluster sizes follow a stretched exponential distribution, and the density of clusters is highest furthest away from the largest (e.g., polar) clusters. Both observations support a model for passive self-assembly rather than active cytoskeletal assembly of the chemotaxis network.

}, keywords = {Bacterial Proteins, Biological, Cell Surface, Chemoreceptor Cells, Chemotaxis, Escherichia coli, Escherichia coli Proteins, Membrane Proteins, Microscopy, Models, Receptors, Recombinant Fusion Proteins, Signal Transduction, Stochastic Processes}, issn = {1545-7885}, doi = {10.1371/journal.pbio.1000137}, url = {http://www.ncbi.nlm.nih.gov/pubmed/19547746}, author = {Greenfield, Derek and McEvoy, Ann L and Shroff, Hari and Crooks, Gavin E and Wingreen, Ned S and Betzig, Eric and Liphardt, Jan} } @article {5363, title = {Three-dimensional nanoscopy of biological samples.}, journal = {2009 Conference on Lasers and Electro-Optics and Quantum Electronics and Laser Science Conference (CLEO/QELS 2009), Vols. 1-5}, volume = {1-5}, year = {2009}, month = {2009 Jun 5}, pages = {147-8}, abstract = {

We have demonstrated super-resolution imaging of protein distributions in cells at depth at multiple layers with a lateral localization precision better than 50 nm. The approach is based on combining photoactivated localization microscopy with temporal focusing.

}, author = {Vaziri, Alipasha and Tang, Jianyong and Shroff, Hari and Shank, Charles V} } @article {4020, title = {Advances in the speed and resolution of light microscopy.}, journal = {Current Opinion in Neurobiology}, volume = {18}, year = {2008}, month = {2008 Dec}, pages = {605-16}, abstract = {

Neurobiological processes occur on spatiotemporal scales spanning many orders of magnitude. Greater understanding of these processes therefore demands improvements in the tools used in their study. Here we review recent efforts to enhance the speed and resolution of one such tool, fluorescence microscopy, with an eye toward its application to neurobiological problems. On the speed front, improvements in beam scanning technology, signal generation rates, and photodamage mediation are bringing us closer to the goal of real-time functional imaging of extended neural networks. With regard to resolution, emerging methods of adaptive optics may lead to diffraction-limited imaging or much deeper imaging in optically inhomogeneous tissues, and super-resolution techniques may prove a powerful adjunct to electron microscopic methods for nanometric neural circuit reconstruction.

}, keywords = {Animals, Electron, Fluorescence, Humans, Microscopy, Nerve Net, Neurology, Photobleaching, Photons}, issn = {1873-6882}, doi = {10.1016/j.conb.2009.03.009}, author = {Ji, Na and Shroff, Hari and Zhong, Haining and Betzig, Eric} } @article {2077, title = {Advances in the speed and resolution of light microscopy. (With commentary)}, journal = {Current Opinion in Neurobiology}, volume = {18}, year = {2008}, month = {2008 Dec}, pages = {605-16}, abstract = {

Neurobiological processes occur on spatiotemporal scales spanning many orders of magnitude. Greater understanding of these processes therefore demands improvements in the tools used in their study. Here we review recent efforts to enhance the speed and resolution of one such tool, fluorescence microscopy, with an eye toward its application to neurobiological problems. On the speed front, improvements in beam scanning technology, signal generation rates, and photodamage mediation are bringing us closer to the goal of real-time functional imaging of extended neural networks. With regard to resolution, emerging methods of adaptive optics may lead to diffraction-limited imaging or much deeper imaging in optically inhomogeneous tissues, and super-resolution techniques may prove a powerful adjunct to electron microscopic methods for nanometric neural circuit reconstruction.

Commentary: A brief review of recent trends in microscopy. The section {\textquotedblleft}Caveats regarding the application of superresolution microscopy{\textquotedblright} was written in an effort to inject a dose of reality and caution into the unquestioning enthusiasm in the academic community for all things superresolution, covering the topics of labeling density and specificity, sample preparation artifacts, speed vs. resolution vs. photodamage, and the implications of signal-to-background for Nyquist vs. Rayleigh definitions of resolution.

}, keywords = {Animals, Electron, Fluorescence, Humans, Microscopy, Nerve Net, Neurology, Photobleaching, Photons}, issn = {1873-6882}, doi = {10.1016/j.conb.2009.03.009}, url = {http://www.ncbi.nlm.nih.gov/pubmed/19375302}, author = {Ji, Na and Shroff, Hari and Zhong, Haining and Betzig, Eric} } @article {2080, title = {High-density mapping of single-molecule trajectories with photoactivated localization microscopy. (With commentary)}, journal = {Nature Methods}, volume = {5}, year = {2008}, month = {2008 Feb}, pages = {155-7}, abstract = {

We combined photoactivated localization microscopy (PALM) with live-cell single-particle tracking to create a new method termed sptPALM. We created spatially resolved maps of single-molecule motions by imaging the membrane proteins Gag and VSVG, and obtained several orders of magnitude more trajectories per cell than traditional single-particle tracking enables. By probing distinct subsets of molecules, sptPALM can provide insight into the origins of spatial and temporal heterogeneities in membranes.

Commentary: As a stepping stone to true live cell PALM (see above), our collaborator Jennifer Lippincott-Schwartz suggested using the sparse photoactivation principle of PALM to track the nanoscale motion of thousands of individual molecules within a single living cell. Termed single particle tracking PALM (sptPALM), Jennifer{\textquoteright}s postdocs Suliana Manley and Jen Gillette used the method in our PALM rig to create spatially resolved maps of diffusion rates in the plasma membrane of live cells. sptPALM is a powerful tool to study the active cytoskeletal or passive diffusional transport of individual molecules with far more measurements per cell than is possible without sparse photoactivation.

}, keywords = {Animals, Cercopithecus aethiops, COS Cells, Fluorescence, Image Enhancement, Membrane Proteins, Microscopy}, issn = {1548-7105}, doi = {10.1038/nmeth.1176}, url = {http://www.ncbi.nlm.nih.gov/pubmed/18193054}, author = {Manley, Suliana and Gillette, Jennifer M and Patterson, George H and Shroff, Hari and Hess, Harald F and Betzig, Eric and Lippincott-Schwartz, Jennifer} } @article {2079, title = {Live-cell photoactivated localization microscopy of nanoscale adhesion dynamics.}, journal = {Nature Methods}, volume = {5}, year = {2008}, month = {2008 May}, pages = {417-23}, abstract = {

We demonstrate live-cell super-resolution imaging using photoactivated localization microscopy (PALM). The use of photon-tolerant cell lines in combination with the high resolution and molecular sensitivity of PALM permitted us to investigate the nanoscale dynamics within individual adhesion complexes (ACs) in living cells under physiological conditions for as long as 25 min, with half of the time spent collecting the PALM images at spatial resolutions down to approximately 60 nm and frame rates as short as 25 s. We visualized the formation of ACs and measured the fractional gain and loss of individual paxillin molecules as each AC evolved. By allowing observation of a wide variety of nanoscale dynamics, live-cell PALM provides insights into molecular assembly during the initiation, maturation and dissolution of cellular processes.

Commentary: The first example of true live cell and time lapse imaging by localization microscopy (as opposed to particle tracking), this paper uses the Nyquist criterion to establish a necessary condition for true spatial resolution based on the density of localized molecules {\textendash} a condition often unmet in claims elsewhere in the superresolution literature.
By any method, higher spatiotemporal resolution requires increasing light exposure at the specimen, making noninvasive imaging increasingly difficult. Here, simultaneous differential interference contrast imaging is used to establish that cells behave physiologically before, during, and after PALM imaging. Similar controls are lacking from many supposed {\textquotedblleft}live cell{\textquotedblright} superresolution demonstrations.

}, keywords = {Animals, Cell Adhesion, Cell Survival, CHO Cells, Computer-Assisted, Cricetinae, Cricetulus, Image Processing, Microscopy, Nanotechnology, Paxillin, Protein Transport, Staining and Labeling}, issn = {1548-7105}, doi = {10.1038/nmeth.1202}, url = {http://www.ncbi.nlm.nih.gov/pubmed/18408726}, author = {Shroff, Hari and Galbraith, Catherine G and Galbraith, James A and Betzig, Eric} } @article {5225, title = {Multilayer three-dimensional super resolution imaging of thick biological samples.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {105}, year = {2008}, month = {2008 Dec 23}, pages = {20221-6}, abstract = {

Recent advances in optical microscopy have enabled biological imaging beyond the diffraction limit at nanometer resolution. A general feature of most of the techniques based on photoactivated localization microscopy (PALM) or stochastic optical reconstruction microscopy (STORM) has been the use of thin biological samples in combination with total internal reflection, thus limiting the imaging depth to a fraction of an optical wavelength. However, to study whole cells or organelles that are typically up to 15 microm deep into the cell, the extension of these methods to a three-dimensional (3D) super resolution technique is required. Here, we report an advance in optical microscopy that enables imaging of protein distributions in cells with a lateral localization precision better than 50 nm at multiple imaging planes deep in biological samples. The approach is based on combining the lateral super resolution provided by PALM with two-photon temporal focusing that provides optical sectioning. We have generated super-resolution images over an axial range of approximately 10 microm in both mitochondrially labeled fixed cells, and in the membranes of living S2 Drosophila cells.

}, keywords = {Animals, Cell Line, Cells, Humans, Imaging, Microscopy, Mitochondria, Organelles, Three-Dimensional}, issn = {1091-6490}, doi = {10.1073/pnas.0810636105}, author = {Vaziri, Alipasha and Tang, Jianyong and Shroff, Hari and Shank, Charles V} } @article {2078, title = {Photoactivated localization microscopy (PALM) of adhesion complexes. (With commentary)}, journal = {Current Protocols in Cell Biology}, volume = {Chapter 4}, year = {2008}, month = {2008 Dec}, pages = {21}, abstract = {

Key to understanding a protein{\textquoteright}s biological function is the accurate determination of its spatial distribution inside a cell. Although fluorescent protein markers allow the targeting of specific proteins with molecular precision, much of this information is lost when the resultant fusion proteins are imaged with conventional, diffraction-limited optics. In response, several imaging modalities that are capable of resolution below the diffraction limit (approximately 200 nm) have emerged. Here, both single- and dual-color superresolution imaging of biological structures using photoactivated localization microscopy (PALM) are described. The examples discussed focus on adhesion complexes: dense, protein-filled assemblies that form at the interface between cells and their substrata. A particular emphasis is placed on the instrumentation and photoactivatable fluorescent protein (PA-FP) tags necessary to achieve PALM images at approximately 20 nm resolution in 5 to 30 min in fixed cells.

Commentary: A paper spearheaded by Hari which gives a thorough description of the methods and hardware needed to successfully practice PALM, including cover slip preparation, cell transfection and fixation, drift correction with fiducials, characterization of on/off contrast ratios for different photoactivted fluorescent proteins, identifying PALM-suitable cells, and mechanical and optical components of a PALM system.

}, keywords = {Animals, Cell Line, Cell-Matrix Junctions, Cells, Fluorescence, Humans, Microscopy, Photobleaching, Transfection}, issn = {1934-2616}, doi = {10.1002/0471143030.cb0421s41}, url = {http://www.ncbi.nlm.nih.gov/pubmed/19085989}, author = {Shroff, Hari and White, Helen and Betzig, Eric} } @article {2082, title = {Dual-color superresolution imaging of genetically expressed probes within individual adhesion complexes. (With commentary)}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {104}, year = {2007}, month = {2007 Dec 18}, pages = {20308-13}, abstract = {

Accurate determination of the relative positions of proteins within localized regions of the cell is essential for understanding their biological function. Although fluorescent fusion proteins are targeted with molecular precision, the position of these genetically expressed reporters is usually known only to the resolution of conventional optics ( approximately 200 nm). Here, we report the use of two-color photoactivated localization microscopy (PALM) to determine the ultrastructural relationship between different proteins fused to spectrally distinct photoactivatable fluorescent proteins (PA-FPs). The nonperturbative incorporation of these endogenous tags facilitates an imaging resolution in whole, fixed cells of approximately 20-30 nm at acquisition times of 5-30 min. We apply the technique to image different pairs of proteins assembled in adhesion complexes, the central attachment points between the cytoskeleton and the substrate in migrating cells. For several pairs, we find that proteins that seem colocalized when viewed by conventional optics are resolved as distinct interlocking nano-aggregates when imaged via PALM. The simplicity, minimal invasiveness, resolution, and speed of the technique all suggest its potential to directly visualize molecular interactions within cellular structures at the nanometer scale.

Commentary: Identifies the photoactivatable fluorescent proteins (PA-FPs) Dronpa and PS-CFP2 as green partners to orange-red PA-FPs such as Kaede and Eos for dual color PALM imaging. Very low crosstalk is demonstrated between the two color channels. Furthermore, since the probes are genetically expressed, they are closely bound to their target proteins and exhibit zero non-specific background. All these properties are essential to unambiguously identify regions of co-localization or separate compartmentalization at the nanoscale, as demonstrated in the examples here.

}, keywords = {Cell Adhesion, Cell Line, Fluorescence, Humans, Light, Luminescent Proteins, Microscopy, Molecular Probes, Nanostructures, Photobleaching, Recombinant Fusion Proteins}, issn = {1091-6490}, doi = {10.1073/pnas.0710517105}, url = {http://www.ncbi.nlm.nih.gov/pubmed/18077327}, author = {Shroff, Hari and Galbraith, Catherine G and Galbraith, James A and White, Helen and Gillette, Jennifer and Olenych, Scott and Davidson, Michael W and Betzig, Eric} }