Advancing cell biology with nanoscale fluorescence imaging: essential practical considerations

Trends in Cell Biology, Год журнала: 2024, Номер 34(8), С. 671 - 684

Опубликована: Янв. 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

Язык: Английский

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Sculpting nuclear envelope identity from the endoplasmic reticulum during the cell cycle

Nucleus, Год журнала: 2024, Номер 15(1)

Опубликована: Янв. 18, 2024

The nuclear envelope (NE) regulates functions, including transcription, nucleocytoplasmic transport, and protein quality control. While the outer membrane of NE is directly continuous with endoplasmic reticulum (ER), has an overall distinct composition from ER, which crucial for its functions. During open mitosis in higher eukaryotes, disassembles during mitotic entry then reforms as a functional territory at end to reestablish compartmentalization. In this review, we examine known mechanisms by reconstitutes ER ER–NE endomembrane system mitosis. Furthermore, based on recent findings indicating that possesses unique lipid metabolism control those explore maintenance identity homeostasis interphase. We also highlight potential significance junctions between NE.

Язык: Английский

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Phenotype structuring in collective cell migration: a tutorial of mathematical models and methods

Journal of Mathematical Biology, Год журнала: 2025, Номер 90(6)

Опубликована: Май 16, 2025

Populations are heterogeneous, deviating in numerous ways. Phenotypic diversity refers to the range of traits or characteristics across a population, where for cells this could be levels signalling, movement and growth activity, etc. Clearly, phenotypic distribution -- how changes over time space major determinant population-level dynamics. For instance, cancerous variations movement, growth, ability evade death may determine its trajectory response therapy. In review, we discuss classical partial differential equation (PDE) approaches modelling cellular systems collective cell migration can extended include structuring. The resulting non-local models which refer as phenotype-structured integro-differential equations (PS-PIDEs) form sophisticated class with rich We set scene through brief history structured population modelling, then review extension several classic including Fisher-KPP Keller-Segel into PS-PIDE form. proceed tutorial-style section on derivation, analysis, simulation techniques. First, show method formally derive these from underlying agent-based models. Second, recount travelling waves PDE spatial spread dynamics concentration phenomena evolutionary dynamics, combine two deduce structuring Third, numerical methods simulate PS-PIDEs, illustrating simple scheme based lines noting finer points consideration. conclude discussion future mathematical challenges.

Язык: Английский

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Optical Microscopy and the Extracellular Matrix Structure: A Review

Cells, Год журнала: 2021, Номер 10(7), С. 1760 - 1760

Опубликована: Июль 12, 2021

Biological tissues are not uniquely composed of cells. A substantial part their volume is extracellular space, which primarily filled by an intricate network macromolecules constituting the matrix (ECM). The ECM serves as scaffolding for and organs throughout body, playing essential role in structural functional integrity. Understanding intimate interaction between cells microenvironment central to our understanding factors driving formation normal versus remodelled tissue, including processes involved chronic fibrotic diseases. visualization a key factor track such changes successfully. This review focused on presenting several optical imaging microscopy modalities used characterize different components. In this review, we describe provide examples applications vast gamut techniques, widefield fluorescence, total internal reflection laser scanning confocal microscopy, multipoint/slit two-photon excited fluorescence (TPEF), second third harmonic generation (SHG, THG), coherent anti-Stokes Raman scattering (CARS), lifetime (FLIM), structured illumination (SIM), stimulated emission depletion (STED), ground-state (GSD), photoactivated localization (PALM/fPALM), well main advantages, limitations.

Язык: Английский

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Quantification of Dendritic Spines Remodeling under Physiological Stimuli and in Pathological Conditions

International Journal of Molecular Sciences, Год журнала: 2021, Номер 22(8), С. 4053 - 4053

Опубликована: Апрель 14, 2021

Numerous brain diseases are associated with abnormalities in morphology and density of dendritic spines, small membranous protrusions whose structural geometry correlates the strength synaptic connections. Thus, quantitative analysis spines remodeling microscopic images is one key elements towards understanding mechanisms neuronal plasticity bases pathology. In following article, we review experimental approaches designed to assess features under physiological stimuli pathological conditions. We compare various methodological pipelines biological models, sample preparation, data analysis, image acquisition, size, statistical analysis. The methodology results relevant experiments systematically summarized a tabular form. particular, focus on regarding number animals, cells, types studied parameters, size observed changes, their significance.

Язык: Английский

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Practical considerations for quantitative light sheet fluorescence microscopy

Nature Methods, Год журнала: 2022, Номер 19(12), С. 1538 - 1549

Опубликована: Окт. 20, 2022

Язык: Английский

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Presynapses contain distinct actin nanostructures

The Journal of Cell Biology, Год журнала: 2023, Номер 222(10)

Опубликована: Авг. 14, 2023

The architecture of the actin cytoskeleton that concentrates at presynapses remains poorly known, hindering our understanding its roles in synaptic physiology. In this work, we measure and visualize presynaptic by diffraction-limited super-resolution microscopy, thanks to a validated model bead-induced cultured neurons. We identify major population actin-enriched more components shows higher vesicle cycling than their non-enriched counterparts. Pharmacological perturbations point an optimal amount presence distinct structures within presynapses. directly these nanostructures using Single Molecule Localization Microscopy (SMLM), defining three types: mesh active zone, rails between zone deeper reserve pools, corrals around whole compartment. Finally, CRISPR-tagging endogenous allows us validate results natural synapses neurons, confirming role enrichment types nanostructures.

Язык: Английский

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Enablers and challenges of spatial omics, a melting pot of technologies

Molecular Systems Biology, Год журнала: 2023, Номер 19(11)

Опубликована: Окт. 16, 2023

Abstract Spatial omics has emerged as a rapidly growing and fruitful field with hundreds of publications presenting novel methods for obtaining spatially resolved information any data type on spatial scales ranging from subcellular to organismal. From technology development perspective, is highly interdisciplinary that integrates imaging omics, molecular analyses, sequencing mass spectrometry, image analysis bioinformatics. The emergence this not only opened window into biology, but also created multiple opportunities, questions, challenges method developers. Here, we provide the perspective developers what makes unique. After providing brief overview state art, discuss technological enablers present our vision about future applications impact melting pot.

Язык: Английский

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The desmosome comes into focus

The Journal of Cell Biology, Год журнала: 2024, Номер 223(9)

Опубликована: Авг. 9, 2024

The desmosome is a cell-cell adhesive junction that provides integrity and mechanical resistance to tissues through its attachment the intermediate filament cytoskeleton. Defects in desmosomes cause diseases impacting heart, epidermis, other epithelia. In this review, we provide historical perspective on discovery of how evolution cellular imaging technologies revealed insights into structure function. We also discuss recent findings using contemporary approaches have informed molecular order, three-dimensional architecture, associations with organelles such as endoplasmic reticulum. Finally, an updated model organization speculate upon novel functions cell signaling center for sensing forms stress.

Язык: Английский

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Image Analysis in Histopathology and Cytopathology: From Early Days to Current Perspectives

Journal of Imaging, Год журнала: 2024, Номер 10(10), С. 252 - 252

Опубликована: Окт. 14, 2024

Both pathology and cytopathology still rely on recognizing microscopical morphologic features, image analysis plays a crucial role, enabling the identification, categorization, characterization of different tissue types, cell populations, disease states within microscopic images. Historically, manual methods have been primary approach, relying expert knowledge experience pathologists to interpret samples. Early were often constrained by computational power complexity biological The advent computers digital imaging technologies challenged exclusivity human eye vision brain skills, transforming diagnostic process in these fields. increasing digitization pathological images has led application more objective efficient computer-aided techniques. Significant advancements brought about integration pathology, machine learning, advanced technologies. continuous progress learning availability data offer exciting opportunities for future. Furthermore, artificial intelligence revolutionized this field, predictive models that assist decision making. future is predicted be marked analysis. promising, will invariably lead enhanced accuracy improved prognostic predictions shape personalized treatment strategies, ultimately leading better patient outcomes.

Язык: Английский

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