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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Lipid Rafts in Signalling, Diseases, and Infections: What Can Be Learned from Fluorescence Techniques?

Membranes, Journal Year: 2025, Volume and Issue: 15(1), P. 6 - 6

Published: Jan. 1, 2025

Lipid rafts are dynamic microdomains in the membrane, rich cholesterol and sphingolipids, that critical for biological processes like cell signalling, membrane trafficking, protein organization. Their essential role is claimed both physiological pathological conditions, including cancer, neurodegenerative diseases, viral infections, making them a key area of research. Fluorescence-based approaches, super-resolution fluorescence microscopy techniques, enable precise analysis organization, dynamics, interactions these microdomains, thanks also to innovative design appropriate fluorescent probes. Moreover, non-invasive approaches allow study live cells, facilitating collection quantitative data under physiologically relevant conditions. This review synthesizes latest insights into lipid underscores how techniques have advanced our understanding microdomains. The findings emphasize pivotal health disease, providing foundation future research potential therapeutic interventions.

Language: Английский

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Phasor-FSTM: a new paradigm for multicolor super-resolution imaging of living cells based on fluorescence modulation and lifetime multiplexing

Light Science & Applications, Journal Year: 2025, Volume and Issue: 14(1)

Published: Jan. 3, 2025

Abstract Multicolor microscopy and super-resolution optical are two widely used techniques that greatly enhance the ability to distinguish resolve structures in cellular imaging. These methods have individually transformed imaging by allowing detailed visualization of subcellular structures, as well organelle interactions. However, integrating multicolor into a single method remains challenging due issues like spectral overlap, crosstalk, photobleaching, phototoxicity, technical complexity. challenges arise from conflicting requirements using different fluorophores for labeling with specific properties We propose novel called phasor-based fluorescence spatiotemporal modulation (Phasor-FSTM). This uses time-resolved detection acquire data encoded photons, employs phasor analysis simultaneously separate multiple components, applies create images. Phasor-FSTM enables identification structural components greater spatial accuracy on an enhanced laser scanning confocal microscope single-wavelength laser. To demonstrate capabilities Phasor-FSTM, we performed two-color four-color at resolution ~λ/5 observed interactions organelles live cells during continuous duration over 20 min. Our stands out its simplicity adaptability, seamlessly fitting existing microscopes without requiring lines excitation, which also provides new avenue other technologies based principles build multi-color systems requirement lower budget.

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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A dynamic role for transcription factors in restoring transcription through mitosis

Biochemical Society Transactions, Journal Year: 2024, Volume and Issue: 52(2), P. 821 - 830

Published: March 25, 2024

Mitosis involves intricate steps, such as DNA condensation, nuclear membrane disassembly, and phosphorylation cascades that temporarily halt gene transcription. Despite this disruption, daughter cells remarkably retain the parent cell's expression pattern, allowing for efficient transcriptional memory after division. Early studies in mammalian suggested transcription factors (TFs) mark genes swift reactivation, a phenomenon termed ‘mitotic bookmarking’, but conflicting data emerged regarding TF presence on mitotic chromosomes. Recent advancements live-cell imaging fixation-free genomics challenge conventional belief universal formaldehyde fixation, revealing dynamic interactions during mitosis. Here, we review recent provide examples of at least four modes TF–DNA interaction mitosis molecular mechanisms govern these interactions. Additionally, explore impact initiation post-mitosis. Taken together, call paradigm shift toward model behavior mitosis, underscoring need incorporating dynamics mechanistic models re-establishing

Language: Английский

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Heterogeneity of late endosome/lysosomes shown by multiplexed DNA-PAINT imaging

The Journal of Cell Biology, Journal Year: 2024, Volume and Issue: 224(1)

Published: Nov. 1, 2024

Late endosomes/lysosomes (LELs) are crucial for numerous physiological processes and their dysfunction is linked to many diseases. Proteomic analyses have identified hundreds of LEL proteins; however, whether these proteins uniformly present on each LEL, or if there cell-type-dependent subpopulations with unique protein compositions unclear. We employed quantitative, multiplexed DNA-PAINT super-resolution imaging examine the distribution seven key (LAMP1, LAMP2, CD63, Cathepsin D, TMEM192, NPC1, LAMTOR4). While LAMP1, D were abundant across LELs, marking a common population, most analyzed associated specific subpopulations. Our approach up eight different based membrane composition. Additionally, our analysis spatial relationships between mitochondria revealed cell-type-specific tendency NPC1-positive LELs be closely positioned mitochondria. will broadly applicable determining organelle heterogeneity single resolution in biological contexts.

Language: Английский

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Microscopy methods for the in vivo study of nanoscale nuclear organization

Biochemical Society Transactions, Journal Year: 2025, Volume and Issue: 53(01)

Published: Feb. 3, 2025

Eukaryotic genomes are highly compacted within the nucleus and organized into complex 3D structures across various genomic physical scales. Organization plays a key role in gene regulation, both facilitating regulatory interactions to promote transcription while also enabling silencing of other genes. Despite functional importance genome organization determining cell identity function, investigating nuclear this wide range scales has been challenging. Microscopy provides opportunity for direct visualization pioneered discoveries field. Nonetheless, nanoscale nucleus, such as nucleosomes chromatin loops, requires super-resolution imaging go beyond ~220 nm diffraction limit. Here, we review recent advances technology their promise uncover new insights at nanoscale. We discuss different modalities how they have applied with focus on light microscopy its application vivo systems. Finally, conclude our perspective continued technical innovations will advance understanding structure function.

Language: Английский

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In-situ microscopy and digital image correlation to study the mechanical characteristics of polymer-based materials

Discover Materials, Journal Year: 2025, Volume and Issue: 5(1)

Published: Feb. 18, 2025

In-situ microscopic methods can help researchers to analyse microstructural changes of materials structures under different conditions (e.g., temperature and pressure) at various length scales. Digital Image Correlation (DIC) combines image registration tracking enable accurate measurements in 2D 3D. This review focuses on combining microscopy DIC study the properties (including natural/synthetic biomaterials, biological samples their composites) academic, public industry settings, including exciting examples bioimaging.

Language: Английский

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Cluster parameter-based DBSCAN maps for image characterization

Computational and Structural Biotechnology Journal, Journal Year: 2025, Volume and Issue: 27, P. 920 - 927

Published: Jan. 1, 2025

Single-molecule localization microscopy techniques are one of the most powerful methods in biological studies, allowing visualization nanoclusters. Cluster analysis algorithms used for quantitative evaluation, with DBSCAN being widely used. Clustering results extremely sensitive to initial parameters; thus, several including maps, have been developed parameter optimization. Here, we introduce cluster parameter-based which directly applicable measured datasets. These maps can be image characterization and optimization through sensitivity studies. We show applicability these simulated datasets compare our recently implemented lacunarity SMLM measurements.

Language: Английский

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Advancing Biosensing through Super-Resolution Fluorescence Microscopy

Biosensors and Bioelectronics, Journal Year: 2025, Volume and Issue: unknown, P. 117374 - 117374

Published: March 1, 2025

Language: Английский

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