Super-resolution microscopy: a brief history and new avenues

Philosophical Transactions of the Royal Society A Mathematical Physical and Engineering Sciences, Journal Year: 2022, Volume and Issue: 380(2220)

Published: Feb. 14, 2022

Super-resolution microscopy (SRM) is a fast-developing field that encompasses fluorescence imaging techniques with the capability to resolve objects below classical diffraction limit of optical resolution. Acknowledged Nobel prize in 2014, numerous SRM methods have meanwhile evolved and are being widely applied biomedical research, all specific strengths shortcomings. While some capable nanometre-scale molecular resolution, others geared towards volumetric three-dimensional multi-colour or fast live-cell imaging. In this editorial review, we pick on latest trends field. We start brief historical overview both conceptual commercial developments. Next, highlight important parameters for successfully particular super-resolution modality. Finally, discuss importance reproducibility quality control significance open-source tools microscopy. This article part Theo Murphy meeting issue 'Super-resolution structured illumination (part 2)'.

Language: Английский

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Medical image super-resolution for smart healthcare applications: A comprehensive survey

Information Fusion, Journal Year: 2023, Volume and Issue: 103, P. 102075 - 102075

Published: Oct. 18, 2023

The digital transformation in healthcare, propelled by the integration of deep learning models and Internet Things (IoT), is creating unprecedented opportunities for improving patient care. However, utilization low-resolution images, often generated IoT devices, introduces biases models, thereby affecting overall clinical decision-making process. While super-resolution techniques have been extensively employed to transform images into high-resolution counterparts, challenge achieving highly accurate image restoration remains unresolved. This especially critical medical imaging domain, where even minor inaccuracies can lead significant model training and, consequently, impact outcomes. Although existing surveys explored various methods their applications across different fields, a comprehensive review emphasizing accuracy its subsequent influence on notably lacking. survey seeks bridge this gap offering systematic current state-of-the-art highlighting limitations surveys, underscoring open questions that merit further research. Specifically, we delve intricacies restoration, identify research gaps unmet challenges optimal emphasize crucial role developing more precise resilient enhance quality performance healthcare applications. Ultimately, fosters deeper comprehension prevailing unresolved field, thus setting stage future efforts focused refining subsequently, boosting efficacy healthcare.

Language: Английский

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RNA granules: functional compartments or incidental condensates?

Genes & Development, Journal Year: 2023, Volume and Issue: 37(9-10), P. 354 - 376

Published: May 1, 2023

RNA granules are mesoscale assemblies that form in the absence of limiting membranes. contain factors for biogenesis and turnover often assumed to represent specialized compartments biochemistry. Recent evidence suggests assemble by phase separation subsoluble ribonucleoprotein (RNP) complexes partially demix from cytoplasm or nucleoplasm. We explore possibility some nonessential condensation by-products arise when RNP exceed their solubility limit as a consequence cellular activity, stress, aging. describe use evolutionary mutational analyses single-molecule techniques distinguish functional "incidental condensates."

Language: Английский

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Advancing cell biology with nanoscale fluorescence imaging: essential practical considerations

Trends in Cell Biology, Journal Year: 2024, Volume and Issue: 34(8), P. 671 - 684

Published: Jan. 5, 2024

Here, we provide an overview of the key factors to consider when using fluorescence nanoscopy (FN), including biological questions that can be addressed and aspects might improve reliability effectiveness FN experiments.We cover main related sample preparation, selection appropriate fixation, affinity-based labels, fluorescent dyes.We discuss current limitations possible future developments in field would facilitate a broader application FN.We multiplexing possibilities (allowing simultaneous detection multiple targets single experiment), live cell imaging for study cellular molecular dynamic processes, quantitative workflows. Recently, biologists have gained access several far-field (FN) technologies allow observation components with ~20 nm resolution. is revolutionizing biology by enabling visualization previously inaccessible subcellular details. While technological advances microscopy are critical field, optimal preparation labeling equally important often overlooked experiments. In this review, methodological experimental must considered performing FN. We present concepts dyes, multiplexing, approaches, microscopy. Consideration these greatly enhances FN, making it exquisite tool numerous applications. Conventional microscopy, widefield confocal has been essential studying morphology composition various organelles localization molecules. The resolution (see Glossary) techniques, usually above ~200 nm, limit macromolecular arrangements nanoscale structures. processes (<20 nm) major step forward because bridges world macromolecules. This effort facilitated recent decades use electron (EM), which provides morphological information at level [1.Winey M. et al.Conventional transmission microscopy.Mol. Biol. Cell. 2014; 25: 319-323Crossref PubMed Google Scholar] expenses limited identifications lack applicability. These overcome achieved resolving capabilities progressively reaching those attained EM. Several approaches based on enable researchers address <20 were difficult answer classical Paradigmatic examples applications include characterization periodicity actin rings synaptic sites neurons, structure nuclear pore complexes, maturation viral particles, organization mitochondrial cristae, mechanisms apoptosis, functioning signaling pathways [2.Fornasiero E.F. Opazo F. Super-resolution biologists.BioEssays. 2015; 37: 436-451Crossref Scholar]. Recent studies reached extremely high resolutions [3.Reinhardt S.C.M. al.Ångström-resolution microscopy.Nature. 2023; 617: 711-716Crossref Scopus (21) even enabled follow events such as stepping behavior kinesin vitro cells [4.Wolff J.O. al.MINFLUX dissects unimpeded walking kinesin-1.Science. 379: 1004-1010Crossref (20) Scholar,5.Deguchi T. al.Direct motor protein living MINFLUX.Science. 1010-1015Crossref (22) render individual molecules, GABA receptors, detail almost comparable cryo-EM [6.Shaib A.H. al.Visualizing proteins expansion microscopy.bioRxiv. (Published online March 10, 2023. https://doi.org/10.1101/2022.08.03.502284)Google compendium summarizes practical keep mind harnessing power nanoscopic biology. By bridging theory practice, roadmap researchers, equipping them know-how successfully navigate intricacies implementing, executing, deriving meaningful data from design experiments starts most suitable technique, each its own specific strengths (Figure 1A ; reviews different see [7.Jacquemet G. al.The biologist's guide super-resolution microscopy.J. Cell Sci. 2020; 133jcs240713Crossref (32) Scholar,8.Bond C. al.Technological processes.Mol. 2022; 82: 315-332Abstract Full Text PDF (25) Scholar]). Two strategies currently able reliably <20-nm samples: camera-based single-molecule (SMLM) [9.Rust M.J. al.Sub-diffraction-limit stochastic optical reconstruction (STORM).Nat. Methods. 2006; 3: 793-796Crossref (6072) minimal photon fluxes (MINFLUX) [10.Balzarotti al.Nanometer tracking molecules fluxes.Science. 2017; 355: 606-612Crossref (657) Based utilized perform on–off switching fluorophores required obtain super-resolved image, SMLM names; example, (STORM), photoactivated (PALM), DNA-points accumulation topography (DNA-PAINT) [11.Jungmann R. al.Single-molecule kinetics transient binding DNA origami.Nano Lett. 2010; 10: 4756-4761Crossref (576) addition two strategies, depending exact settings, stimulated emission depletion (STED) [12.Hell S.W. Wichmann J. Breaking diffraction emission: stimulated-emission-depletion microscopy.Opt. 1994; 19: 780Crossref achieve ~50 below, although laser not compatible conventional samples. technologies, approach aimed enlarging sample, then imaged either diffraction-limited or [13.Chen al.Optical imaging. Expansion microscopy.Science. 347: 543-548Crossref (858) With exception microcopy, specimens, opening avenue understanding dynamics native conditions (Box 1). Overall, four potential uncover as-yet unexplored detail. At same time, their capability requires protocols introduction artifacts tools linkage errors.Box 1Nanometer-scale cells: there yet?FN visualizes entire structures over time 'whole', allowing changes, recommended endoplasmic reticulum, chemical fixation introduces visible [19.Hoffman D.P. al.Correlative three-dimensional block-face whole vitreously frozen cells.Science. 367eaaz5357Crossref (33) our opinion, least connected implementation detailed herein.PhototoxicityPhototoxicity arises absorbed light generates free radicals reactive oxygen species, ultimately causing genomic damage, stress, degradation Figure 1D text). Light both endogenous additionally increase local temperature sample. To some effects, combinations image adaptive illumination [75.Heine al.Adaptive-illumination STED nanoscopy.Proc. Natl. Acad. U. S. A. 114: 9797-9802Crossref (0) Scholar], event-triggered emerging [76.Alvelid al.Event-triggered imaging.Nat. 1268-1275Crossref (15) Furthermore, novel fluorophore classes self-blinking dyes red photoswitchable exist, do require blue excited lower doses [77.Pennacchietti al.Fast reversibly photoswitching live-cell RESOLFT nanoscopy.Nat. 2018; 15: 601-604Crossref (58) Scholar].Low acquisition frequencyLow frequency resolution, but compromises interpretation fast processes. SMLM, limiting factor sufficient number while, scanning-based steps brightness size view. Reducing view spent pixel (dwell time) speed up imaging, decreased contextual SNR. will strongly benefit parallelization deep learning temporal performances.Availability impact cellsThe availability cannot ignored. Some probes drugs bind target affinity, interfering physiology targeted molecule (e.g., phalloidin). An alternative genetically encoded tags or, better, combined genome-editing [78.Bottanelli al.A physiological role ARF1 formation bidirectional tubules Golgi.Mol. 28: 1676-1687Crossref Table 1 text).Imaging depth large viewImaging many samples monolayers. Although feasibility specialized demonstrated [79.Kim al.Oblique-plane tissues small intact animals.Nat. 2019; 16: 853-857Crossref (54) deeper than 10–50 μm methods challenging. engineered illumination, optics, restoration algorithms, multiphoton excitation.Ultimately, best technology selected setup precise question being addressed, controls phototoxic cause damage considered. should done settings less perturbation troubleshooting conditions. For far-red commonly used compared 405-laser irradiance. herein. Phototoxicity Low performances. Availability Imaging excitation. Ultimately, short 'no', all problems solved. Careful consideration given whether necessary questions. determine interest (POI) localized lysosomes mitochondria, POI outer membrane inner matrix, apart. general, strength increased precision biomolecules 1B,C). A simple rule thumb decide needed understand spatial order tens nanometers allows formulation fundamentally hypotheses process under investigation. serendipitous observations made and, cases, revealed new observable [14.Xu K. al.Actin, spectrin, associated form periodic cytoskeletal axons.Science. 2013; 339: 452-456Crossref (888) reason, exploratory Very high-resolution informative, could ignored become challenges Live ultimate goal studies, still due phototoxicity early stage foreseeable relevant near future. fixed already changing field; therefore, concentrate here specimens 1D–F). workflow 'non-live' first optimized avoid artifacts. After antibodies, reveal identities position reporter proximity. relative absolute biologically numbers images (quantitative FN), further measures considerations taken. Box 2, review issues explain why density, stoichiometry, error context. Finally, 3, conditions.Box 2Quantitative FN: versus observed moleculesQuantitative detected matches (or, more precisely, correlates) real Therefore, prerequisites controlled efficiency ability detect fluorophores.A monovalent affinity carrying (or of) reporting 2A Moreover, utilize covalently linked ensure stably labeled preferred. multivalent polyclonal reagents stochastically secondary antibodies approximately one six fluorophores) avoided since correlation between reporters inconsistent.Even ideal case decorated label, possibility exists only decorating label functional. Indeed, inactivated, damaged, during procedure 1E Detecting densely challenging, problem known crowding. When distances single-digit nanometer scale, photophysical interactions occur, resulting undesired alterations properties. happens structure, Förster resonance energy transfer (FRET) H-dimer if separated molecular-scale [80.Ogawa al.H-Type dimer fluorophores: mechanism activatable, vivo imaging.ACS Chem. 2009; 4: 535-546Crossref (153) Notably, reported antibody [81.Helmerich D.A. al.Multiple-labeled behave like emitters buffer.ACS Nano. 14: 12629-12641Crossref (13) serving 'super emitter' while others remain dark state. crowding reversed, physically creating distance Other DNA-PAINT, deal differently modulating concentration probe light-controllable [82.Raymo F.M. Photoactivatable synthetic nanoscale.J. Phys. 2012; 2379-2385Crossref (60) 2D text).While quantification easily achieved, molecule-counting proposed comprehensive recently published challenge [83.Hugelier al.Quantitative microscopy.Annu. Rev. Biophys. 52: 139-160Crossref (1) Importantly, need calibration benchmarked against markers biochemically western blot, liquid chromatography, mass spectrometry).Box 3Fluorophore no one-size-fits-all solutionsA variety developed fulfill requirement technique (Table I). Factors selecting charge, quantum yield, photostability 2C photostable fluorophores, MINFLUX rely switch non-emitting emitting states. mechanisms, refer reader text.The scaffolds cyanines rhodamines. Among cyanines, Alexa Fluor 647 gold standard blinks presence reducing agents UV [84.Berlier J.E. comparison long-wavelength Cy Dyes: bioconjugates.J. Histochem. Cytochem. 2003; 51: 1699-1712Crossref (229) Rhodamines relatively modified tune spectral properties [85.Grimm J.B. general method optimize functionalize red-shifted rhodamine dyes.Nat. 17: 815-821Crossref (85) permeability [86.Lukinavičius near-infrared proteins.Nat. 5: 132-139Crossref (648) equilibrium open closed non-fluorescent forms. regulation latter induces cell-compatible spontaneous blinking Scholar].Other frequently coumarin, oxazine, BODIPY 2E Coumarins among smallest generate variants Stokes shift, advantageous low background [87.Nizamov al.Phosphorylated 3-heteroarylcoumarins nanoscopy.Chem. Eur. 18: 16339-16348Crossref (45) Oxazines [88.Wombacher al.Live-cell trimethoprim conjugates.Nat. 7: 717-719Crossref (282) absorbance, extinction coefficient, 'blink' buffers containing oxidizing agents. valued sharp absorption spectra very yield coefficient [89.Kowada al.BODIPY-based cells.Chem. Soc. 44: 4953-4972Crossref highly hydrophobic nature poor off-switching properties, light-dose photoactivatable make attractive [90.Wijesooriya C.S. localization-based imaging.Angew. Int. Ed. 57 (12685–1268)Crossref (74) Scholar].In after identified, always considering specifications available instrument lasers detectors). choice also driven needs, multicolor autofluorescence reduce background, counting naked may themselves affinities lipophilic stain membranes). evaluated designing experiments, (reporters) without targeting moiety whenever 2B recommend inexperienced users consult expert select dye application.Table IProperties dyesFluorophore classCoumarinsRhodaminesCyaninesBODIPYsOxazinesCommercial examplesAlexa 350, Pacific BlueAlexa 488, silicon-rhodamine, TMRAlexa 647, 555, Cy5BODIPY FL, TMRAtto 655, Atto 680Spectral range (nm)360–700500–750500–1000500–700600–750Extinction (cm–1M–1)15 000–60 00080 000–150 000130 000–250 00060 000–100 000110 000–130 000Quantum yield0.4–0.90.1–0.90.1–0.60.8–0.90.1–0.6Photostability+++++++++++Compatibility methodsSTED, SMLMSTED, MINFLUX, microscopySTED, SMLMSMLM Open table tab Quantitative fluorophores. inconsistent. Even sin

Language: Английский

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Insight into the Functional Dynamics and Challenges of Exosomes in Pharmaceutical Innovation and Precision Medicine

Pharmaceutics, Journal Year: 2024, Volume and Issue: 16(6), P. 709 - 709

Published: May 24, 2024

Of all the numerous nanosized extracellular vesicles released by a cell, endosomal-originated exosomes are increasingly recognized as potential therapeutics, owing to their inherent stability, low immunogenicity, and targeted delivery capabilities. This review critically evaluates transformative of exosome-based modalities across pharmaceutical precision medicine landscapes. Because precise biomolecular cargo delivery, posited ideal candidates in drug enhancing regenerative strategies, advancing diagnostic technologies. Despite significant market growth projections exosome therapy, its utilization is encumbered substantial scientific regulatory challenges. These include lack universally accepted protocols for isolation complexities associated with navigating environment, particularly guidelines set forth U.S. Food Drug Administration (FDA). presents comprehensive overview current research trajectories aimed at addressing these impediments discusses prospective advancements that could substantiate clinical translation exosomal therapies. By providing analysis both capabilities hurdles therapeutic applications, this article aims inform direct future paradigms, thereby fostering integration systems into mainstream practice.

Language: Английский

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Visualizing proteins by expansion microscopy

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2022, Volume and Issue: unknown

Published: Aug. 5, 2022

Abstract Fluorescence imaging is one of the most versatile and widely-used tools in biology 1 . Although techniques to overcome diffraction barrier were introduced more than two decades ago, nominal attainable resolution kept improving 2, 3 , fluorescence microscopy still fails image morphology single proteins or small molecular complexes, either purified a cellular context 4, 5 Here we report solution this problem, form o ne-step n anoscale e xpansion (ONE) microscopy. We combined 10-fold axial expansion specimen (1000-fold by volume) with fluctuation analysis 6, 7 enable description cultured cells, tissues, viral particles, complexes proteins. At level, using immunostaining, our technology revealed detailed nanoscale arrangements synaptic proteins, including quasi-regular organisation PSD95 clusters. molecule upon main chain fluorescent labelling, could visualise shape individual membrane soluble Moreover, conformational changes undergone ∼17 kDa protein calmodulin Ca 2+ binding readily observable. also imaged classified aggregates cerebrospinal fluid samples from Parkinson’s Disease (PD) patients, which represents promising new development towards improved PD diagnosis. ONE compatible conventional microscopes can be performed software provide here as free, open-source package. This bridges gap between high-resolution structural light microscopy, provides avenue for discoveries medicine.

Language: Английский

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Resolution enhancement with deblurring by pixel reassignment

Advanced Photonics, Journal Year: 2023, Volume and Issue: 5(06)

Published: Oct. 27, 2023

Improving the spatial resolution of a fluorescence microscope has been an ongoing challenge in imaging community. To address this challenge, variety approaches have taken, ranging from instrumentation development to image postprocessing. An example latter is deconvolution, where images are numerically deblurred based on knowledge point spread function. However, deconvolution can easily lead noise-amplification artifacts. Deblurring by postprocessing also negativities or fail conserve local linearity between sample and image. We describe here simple deblurring algorithm pixel reassignment that inherently avoids such artifacts be applied general modalities fluorophore types. Our helps distinguish nearby fluorophores, even when these separated distances smaller than conventional limit, helping facilitate, for example, application single-molecule localization microscopy dense samples. demonstrate versatility performance our under conditions.

Language: Английский

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The membrane surface as a platform that organizes cellular and biochemical processes

Developmental Cell, Journal Year: 2023, Volume and Issue: 58(15), P. 1315 - 1332

Published: July 6, 2023

Membranes are essential for life. They act as semi-permeable boundaries that define cells and organelles. In addition, their surfaces actively participate in biochemical reaction networks, where they confine proteins, align partners, directly control enzymatic activities. Membrane-localized reactions shape cellular membranes, the identity of organelles, compartmentalize processes, can even be source signaling gradients originate at plasma membrane reach into cytoplasm nucleus. The surface is, therefore, an platform upon which myriad processes scaffolded. this review, we summarize our current understanding biophysics biochemistry membrane-localized with particular focus on insights derived from reconstituted systems. We discuss how interplay factors results self-organization, condensation, assembly, activity, emergent properties them.

Language: Английский

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Quantitative Single-Molecule Localization Microscopy

Annual Review of Biophysics, Journal Year: 2023, Volume and Issue: 52(1), P. 139 - 160

Published: May 9, 2023

Super-resolution fluorescence microscopy allows the investigation of cellular structures at nanoscale resolution using light. Current developments in super-resolution have focused on reliable quantification underlying biological data. In this review, we first describe basic principles techniques such as stimulated emission depletion (STED) and single-molecule localization (SMLM), then give a broad overview methodological to quantify data, particularly those geared toward SMLM We cover commonly used spatial point pattern analysis, colocalization, protein copy number but also more advanced structural modeling, single-particle tracking, biosensing. Finally, provide an outlook exciting new research directions which quantitative might be applied.

Language: Английский

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Expansion-enhanced super-resolution radial fluctuations enable nanoscale molecular profiling of pathology specimens

Nature Nanotechnology, Journal Year: 2023, Volume and Issue: 18(4), P. 336 - 342

Published: April 1, 2023

Expansion microscopy physically enlarges biological specimens to achieve nanoscale resolution using diffraction-limited systems1. However, optimal performance is usually reached laser-based systems (for example, confocal microscopy), restricting its broad applicability in clinical pathology, as most centres have access only light-emitting diode (LED)-based widefield systems. As a possible alternative, computational method for image enhancement, namely, super-resolution radial fluctuations (SRRF)2,3, has recently been developed. this not explored pathology date, because on own, it does sufficient routine use. Here, we report expansion-enhanced (ExSRRF), simple, robust, scalable and accessible workflow that provides of up 25 nm LED-based microscopy. ExSRRF enables molecular profiling subcellular structures from archival formalin-fixed paraffin-embedded tissues complex experimental specimens, including ischaemic, degenerative, neoplastic, genetic immune-mediated disorders. Furthermore, examples potential application show can be used identify quantify classical features endoplasmic reticulum stress the murine ischaemic kidney diagnostic ultrastructural human biopsies.

Language: Английский

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