Democratising deep learning for microscopy with ZeroCostDL4Mic

Nature Communications, Journal Year: 2021, Volume and Issue: 12(1)

Published: April 15, 2021

Abstract Deep Learning (DL) methods are powerful analytical tools for microscopy and can outperform conventional image processing pipelines. Despite the enthusiasm innovations fuelled by DL technology, need to access compatible resources train networks leads an accessibility barrier that novice users often find difficult overcome. Here, we present ZeroCostDL4Mic, entry-level platform simplifying leveraging free, cloud-based computational of Google Colab. ZeroCostDL4Mic allows researchers with no coding expertise apply key perform tasks including segmentation (using U-Net StarDist), object detection YOLOv2), denoising CARE Noise2Void), super-resolution Deep-STORM), image-to-image translation Label-free prediction - fnet, pix2pix CycleGAN). Importantly, provide suitable quantitative each network evaluate model performance, allowing optimisation. We demonstrate application study multiple biological processes.

Language: Английский

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Focal adhesion dynamics in cellular function and disease

Cellular Signalling, Journal Year: 2021, Volume and Issue: 85, P. 110046 - 110046

Published: May 15, 2021

Language: Английский

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iU-ExM: nanoscopy of organelles and tissues with iterative ultrastructure expansion microscopy

Nature Communications, Journal Year: 2023, Volume and Issue: 14(1)

Published: Nov. 30, 2023

Expansion microscopy (ExM) is a highly effective technique for super-resolution fluorescence that enables imaging of biological samples beyond the diffraction limit with conventional microscopes. Despite development several enhanced protocols, ExM has not yet demonstrated ability to achieve precision nanoscopy techniques such as Single Molecule Localization Microscopy (SMLM). Here, address this limitation, we have developed an iterative ultrastructure expansion (iU-ExM) approach achieves SMLM-level resolution. With iU-ExM, it now possible visualize molecular architecture gold-standard samples, eight-fold symmetry nuclear pores or organization conoid in Apicomplexa. its wide-ranging applications, from isolated organelles cells and tissue, iU-ExM opens new avenues scientists studying structures functions.

Language: Английский

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Bright and stable monomeric green fluorescent protein derived from StayGold

Nature Methods, Journal Year: 2024, Volume and Issue: 21(4), P. 657 - 665

Published: Feb. 26, 2024

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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Quantification of tumor heterogeneity: from data acquisition to metric generation

Trends in biotechnology, Journal Year: 2021, Volume and Issue: 40(6), P. 647 - 676

Published: Dec. 28, 2021

Tumors are unique and complex ecosystems, in which heterogeneous cell subpopulations with variable molecular profiles, aggressiveness, proliferation potential coexist interact. Understanding how heterogeneity influences tumor progression has important clinical implications for improving diagnosis, prognosis, treatment response prediction. Several recent innovations data acquisition methods computational metrics have enabled the quantification of spatiotemporal across different scales organization. Here, we summarize most promising efforts from a common experimental perspective, discussing their advantages, shortcomings, challenges. With personalized medicine entering new era unprecedented opportunities, our vision is that future workflows integrating modalities, scales, dimensions to capture intricate aspects ecosystem open avenues improved patient care.

Language: Английский

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Unravelling cell migration: defining movement from the cell surface

Cell Adhesion & Migration, Journal Year: 2022, Volume and Issue: 16(1), P. 25 - 64

Published: May 1, 2022

Cell motility is essential for life and development. Unfortunately, cell migration also linked to several pathological processes, such as cancer metastasis. Cells' ability migrate relies on many actors. Cells change their migratory strategy based phenotype the properties of surrounding microenvironment. is, therefore, an extremely complex phenomenon. Researchers have investigated more than a century. Recent discoveries uncovered some mysteries associated with mechanisms involved in migration, intracellular signaling mechanics. These findings involve different players, including transmembrane receptors, adhesive complexes, cytoskeletal components , nucleus, extracellular matrix. This review aims give global overview our current understanding migration.

Language: Английский

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Extending resolution within a single imaging frame

Nature Communications, Journal Year: 2022, Volume and Issue: 13(1)

Published: Dec. 2, 2022

Abstract The resolution of fluorescence microscopy images is limited by the physical properties light. In last decade, numerous super-resolution (SRM) approaches have been proposed to deal with such hindrance. Here we present Mean-Shift Super Resolution (MSSR), a new SRM algorithm based on Mean Shift theory, which extends spatial single beyond diffraction limit MSSR works low and high fluorophore densities, not architecture optical setup applicable as well temporal series. theoretical resolution, optimized real-world imaging conditions analysis image stacks, has measured be 40 nm. Furthermore, denoising capabilities that outperform other approaches. Along its wide accessibility, powerful, flexible, generic tool for multidimensional live cell applications.

Language: Английский

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High-fidelity 3D live-cell nanoscopy through data-driven enhanced super-resolution radial fluctuation

Nature Methods, Journal Year: 2023, Volume and Issue: 20(12), P. 1949 - 1956

Published: Nov. 13, 2023

Abstract Live-cell super-resolution microscopy enables the imaging of biological structure dynamics below diffraction limit. Here we present enhanced radial fluctuations (eSRRF), substantially improving image fidelity and resolution compared to original SRRF method. eSRRF incorporates automated parameter optimization based on data itself, giving insight into trade-off between fidelity. We demonstrate across a range modalities systems. Notably, extend three dimensions by combining it with multifocus microscopy. This realizes live-cell volumetric an acquisition speed ~1 volume per second. provides accessible approach, maximizing information extraction varied experimental conditions while minimizing artifacts. Its optimal prediction strategy is generalizable, moving toward unbiased optimized analyses in

Language: Английский

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Imagining the future of optical microscopy: everything, everywhere, all at once

Communications Biology, Journal Year: 2023, Volume and Issue: 6(1)

Published: Oct. 28, 2023

The optical microscope has revolutionized biology since at least the 17

Language: Английский

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