Advancing cell biology with nanoscale fluorescence imaging: essential practical considerations
2024; Elsevier BV; Volume: 34; Issue: 8 Linguagem: Inglês
10.1016/j.tcb.2023.12.001
ISSN1879-3088
AutoresElisa D’Este, Gražvydas Lukinavičius, Richard Lincoln, Felipe Opazo, Eugenio F. Fornasiero,
Tópico(s)Cell Image Analysis Techniques
ResumoHere, we provide an overview of the key factors to consider when using fluorescence nanoscopy (FN), including biological questions that can be addressed and aspects that might improve the reliability and effectiveness of FN experiments.We cover the main aspects related to sample preparation, including the selection of appropriate fixation, affinity-based labels, and fluorescent dyes.We discuss current limitations and possible future developments in the field that would facilitate a broader application of FN.We discuss multiplexing possibilities (allowing the simultaneous detection of multiple targets in a single experiment), live cell imaging for the study of cellular and molecular dynamic processes, and quantitative workflows. Recently, biologists have gained access to several far-field fluorescence nanoscopy (FN) technologies that allow the observation of cellular components with ~20 nm resolution. FN is revolutionizing cell biology by enabling the visualization of previously inaccessible subcellular details. While technological advances in microscopy are critical to the field, optimal sample preparation and labeling are equally important and often overlooked in FN experiments. In this review, we provide an overview of the methodological and experimental factors that must be considered when performing FN. We present key concepts related to the selection of affinity-based labels, dyes, multiplexing, live cell imaging approaches, and quantitative microscopy. Consideration of these factors greatly enhances the effectiveness of FN, making it an exquisite tool for numerous biological applications. Recently, biologists have gained access to several far-field fluorescence nanoscopy (FN) technologies that allow the observation of cellular components with ~20 nm resolution. FN is revolutionizing cell biology by enabling the visualization of previously inaccessible subcellular details. While technological advances in microscopy are critical to the field, optimal sample preparation and labeling are equally important and often overlooked in FN experiments. In this review, we provide an overview of the methodological and experimental factors that must be considered when performing FN. We present key concepts related to the selection of affinity-based labels, dyes, multiplexing, live cell imaging approaches, and quantitative microscopy. Consideration of these factors greatly enhances the effectiveness of FN, making it an exquisite tool for numerous biological applications. Conventional fluorescence microscopy, including widefield and confocal microscopy, has been essential for studying the morphology and composition of various cellular organelles and the localization of molecules. The resolution (see Glossary) of these techniques, usually above ~200 nm, is a limit for the study of macromolecular arrangements and nanoscale structures. The study of biological processes with nanoscale resolution (<20 nm) is a major step forward for microscopy, because it bridges the world of subcellular biology with that of macromolecules. This effort has been greatly facilitated in recent decades by the use of electron microscopy (EM), which provides exquisite morphological information at the molecular level [1.Winey M. et al.Conventional transmission electron microscopy.Mol. Biol. Cell. 2014; 25: 319-323Crossref PubMed Google Scholar] at the expenses of limited molecular identifications and lack of live cell applicability. These limitations are overcome by fluorescent microscopy, the achieved resolving capabilities of which are progressively reaching those attained by EM. Several recent approaches based on FN enable researchers to address questions at <20 nm that were difficult to answer with classical fluorescence microscopy. Paradigmatic examples of FN applications include the characterization of the periodicity of actin rings and synaptic sites in neurons, the structure of nuclear pore complexes, the maturation of viral particles, the organization of mitochondrial cristae, the mechanisms of apoptosis, and the functioning of signaling pathways [2.Fornasiero E.F. Opazo F. Super-resolution imaging for cell biologists.BioEssays. 2015; 37: 436-451Crossref PubMed Google Scholar]. Recent FN studies have reached extremely high resolutions [3.Reinhardt S.C.M. et al.Ångström-resolution fluorescence microscopy.Nature. 2023; 617: 711-716Crossref PubMed Scopus (21) Google Scholar] and even enabled researchers to follow molecular events such as the stepping behavior of kinesin in vitro and in cells [4.Wolff J.O. et al.MINFLUX dissects the unimpeded walking of kinesin-1.Science. 2023; 379: 1004-1010Crossref Scopus (20) Google Scholar,5.Deguchi T. et al.Direct observation of motor protein stepping in living cells using MINFLUX.Science. 2023; 379: 1010-1015Crossref Scopus (22) Google Scholar] and to render the structure of individual molecules, such as GABA receptors, with a level of detail almost comparable to that of cryo-EM [6.Shaib A.H. et al.Visualizing proteins by expansion microscopy.bioRxiv. 2023; (Published online March 10, 2023. https://doi.org/10.1101/2022.08.03.502284)Google Scholar]. In this review, we present a compendium that summarizes practical aspects to keep in mind for harnessing the power of nanoscopic imaging in the field of cell biology. By bridging theory and practice, we provide a roadmap for researchers, equipping them with the essential know-how to successfully navigate the intricacies of implementing, executing, and deriving meaningful data from FN experiments. The design of FN experiments starts from the selection of the most suitable microscopy technique, each with its own specific limitations and strengths (Figure 1A ; for recent reviews of the selection of different microscopy approaches, see [7.Jacquemet G. et al.The cell biologist's guide to super-resolution microscopy.J. Cell Sci. 2020; 133jcs240713Crossref PubMed Scopus (32) Google Scholar,8.Bond C. et al.Technological advances in super-resolution microscopy to study cellular processes.Mol. Cell. 2022; 82: 315-332Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar]). Two major far-field fluorescence microscopy strategies are currently able to reliably provide <20-nm resolution in biological samples: camera-based single-molecule localization microscopy (SMLM) [9.Rust M.J. et al.Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM).Nat. Methods. 2006; 3: 793-796Crossref PubMed Scopus (6072) Google Scholar] and minimal photon fluxes (MINFLUX) [10.Balzarotti F. et al.Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes.Science. 2017; 355: 606-612Crossref PubMed Scopus (657) Google Scholar]. Based on the mechanisms utilized to perform the on–off switching of the fluorophores required to obtain a super-resolved image, camera-based SMLM technologies have different names; for example, stochastic optical reconstruction microscopy (STORM), photoactivated localization microscopy (PALM), and DNA-points accumulation for imaging in topography (DNA-PAINT) [11.Jungmann R. et al.Single-molecule kinetics and super-resolution microscopy by fluorescence imaging of transient binding on DNA origami.Nano Lett. 2010; 10: 4756-4761Crossref PubMed Scopus (576) Google Scholar]. In addition to the above two strategies, depending on the exact imaging settings, stimulated emission depletion (STED) [12.Hell S.W. Wichmann J. Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy.Opt. Lett. 1994; 19: 780Crossref PubMed Google Scholar] can achieve resolutions of ~50 nm and below, although the power of the depletion laser required for FN applications is usually not compatible with conventional biological samples. In addition to these technologies, expansion microscopy is a sample preparation approach aimed at enlarging the sample, which can then be imaged using either conventional diffraction-limited or nanoscopy approaches [13.Chen F. et al.Optical imaging. Expansion microscopy.Science. 2015; 347: 543-548Crossref PubMed Scopus (858) Google Scholar]. With the exception of expansion microcopy, FN technologies are compatible with living specimens, opening the avenue to the understanding of molecular dynamics in native conditions (Box 1). Overall, these four microscopy technologies have the potential to uncover as-yet unexplored biological aspects with exquisite detail. At the same time, their high resolving capability requires the use of sample preparation protocols that limit the introduction of artifacts and the use of labeling tools that have minimal linkage errors.Box 1Nanometer-scale fluorescent imaging in living cells: are we there yet?FN live cell imaging visualizes entire cellular structures over time as a 'whole', allowing detection of morphological changes, and is recommended for organelles such as the endoplasmic reticulum, in which conventional chemical fixation often introduces artifacts that are visible in FN [19.Hoffman D.P. et al.Correlative three-dimensional super-resolution and block-face electron microscopy of whole vitreously frozen cells.Science. 2020; 367eaaz5357Crossref PubMed Scopus (33) Google Scholar]. In our opinion, at least four connected aspects limit the implementation of live FN applications as detailed herein.PhototoxicityPhototoxicity arises when the absorbed light generates free radicals and reactive oxygen species, ultimately causing genomic damage, mitochondrial stress, and protein degradation (see Figure 1D in main text). Light might be absorbed by both endogenous molecules or the fluorophores and can additionally increase the local temperature in the sample. To limit some of these effects, combinations of image reconstruction strategies, adaptive illumination approaches [75.Heine J. et al.Adaptive-illumination STED nanoscopy.Proc. Natl. Acad. Sci. U. S. A. 2017; 114: 9797-9802Crossref PubMed Scopus (0) Google Scholar], and event-triggered approaches are emerging [76.Alvelid J. et al.Event-triggered STED imaging.Nat. Methods. 2022; 19: 1268-1275Crossref PubMed Scopus (15) Google Scholar]. Furthermore, novel fluorophore classes of self-blinking dyes or red photoswitchable proteins exist, which do not require blue light and can be excited with lower light doses [77.Pennacchietti F. et al.Fast reversibly photoswitching red fluorescent proteins for live-cell RESOLFT nanoscopy.Nat. Methods. 2018; 15: 601-604Crossref PubMed Scopus (58) Google Scholar].Low image acquisition frequencyLow image acquisition frequency is often required to increase resolution, but compromises the interpretation of fast biological processes. In SMLM, a limiting factor is the localization of a sufficient number of molecules, while, in scanning-based approaches, the limiting steps are the brightness of the fluorophore and size of the field of view. Reducing the field of view or the time spent on each pixel (dwell time) can speed up the imaging, although at the expenses of decreased contextual information and SNR. FN will strongly benefit from parallelization and the use of deep learning to improve temporal performances.Availability and impact of labeling on living cellsThe availability and impact of labeling on living cells cannot be ignored. Some probes are drugs that bind to their target with high affinity, often interfering with the physiology of the targeted molecule (e.g., phalloidin). An alternative is to use genetically encoded tags or, even better, minimal tags combined with genome-editing approaches [78.Bottanelli F. et al.A novel physiological role for ARF1 in the formation of bidirectional tubules from the Golgi.Mol. Biol. Cell. 2017; 28: 1676-1687Crossref PubMed Google Scholar] (see Table 1 in main text).Imaging depth and large field of viewImaging depth and a large field of view are important because many biological samples are not single cell monolayers. Although its feasibility specialized has been demonstrated [79.Kim J. et al.Oblique-plane single-molecule localization microscopy for tissues and small intact animals.Nat. Methods. 2019; 16: 853-857Crossref PubMed Scopus (54) Google Scholar], imaging deeper than 10–50 μm in both SMLM and STED methods is challenging. The field would benefit from the use of engineered illumination, adaptive optics, image restoration algorithms, and multiphoton excitation.Ultimately, the best FN technology for live cell imaging must be selected based on the experimental setup and the precise biological question being addressed, and it is essential to include controls for possible phototoxic effects, such as conditions that are present that might cause damage to biological processes but are not considered. This should be done by using settings that cause less perturbation of biological conditions and can be achieved by troubleshooting the selection of appropriate labeling strategies and imaging conditions. For example, far-red light commonly used in the STED depletion laser is less phototoxic compared with the 405-laser used for SMLM at the same irradiance. FN live cell imaging visualizes entire cellular structures over time as a 'whole', allowing detection of morphological changes, and is recommended for organelles such as the endoplasmic reticulum, in which conventional chemical fixation often introduces artifacts that are visible in FN [19.Hoffman D.P. et al.Correlative three-dimensional super-resolution and block-face electron microscopy of whole vitreously frozen cells.Science. 2020; 367eaaz5357Crossref PubMed Scopus (33) Google Scholar]. In our opinion, at least four connected aspects limit the implementation of live FN applications as detailed herein. Phototoxicity Phototoxicity arises when the absorbed light generates free radicals and reactive oxygen species, ultimately causing genomic damage, mitochondrial stress, and protein degradation (see Figure 1D in main text). Light might be absorbed by both endogenous molecules or the fluorophores and can additionally increase the local temperature in the sample. To limit some of these effects, combinations of image reconstruction strategies, adaptive illumination approaches [75.Heine J. et al.Adaptive-illumination STED nanoscopy.Proc. Natl. Acad. Sci. U. S. A. 2017; 114: 9797-9802Crossref PubMed Scopus (0) Google Scholar], and event-triggered approaches are emerging [76.Alvelid J. et al.Event-triggered STED imaging.Nat. Methods. 2022; 19: 1268-1275Crossref PubMed Scopus (15) Google Scholar]. Furthermore, novel fluorophore classes of self-blinking dyes or red photoswitchable proteins exist, which do not require blue light and can be excited with lower light doses [77.Pennacchietti F. et al.Fast reversibly photoswitching red fluorescent proteins for live-cell RESOLFT nanoscopy.Nat. Methods. 2018; 15: 601-604Crossref PubMed Scopus (58) Google Scholar]. Low image acquisition frequency Low image acquisition frequency is often required to increase resolution, but compromises the interpretation of fast biological processes. In SMLM, a limiting factor is the localization of a sufficient number of molecules, while, in scanning-based approaches, the limiting steps are the brightness of the fluorophore and size of the field of view. Reducing the field of view or the time spent on each pixel (dwell time) can speed up the imaging, although at the expenses of decreased contextual information and SNR. FN will strongly benefit from parallelization and the use of deep learning to improve temporal performances. Availability and impact of labeling on living cells The availability and impact of labeling on living cells cannot be ignored. Some probes are drugs that bind to their target with high affinity, often interfering with the physiology of the targeted molecule (e.g., phalloidin). An alternative is to use genetically encoded tags or, even better, minimal tags combined with genome-editing approaches [78.Bottanelli F. et al.A novel physiological role for ARF1 in the formation of bidirectional tubules from the Golgi.Mol. Biol. Cell. 2017; 28: 1676-1687Crossref PubMed Google Scholar] (see Table 1 in main text). Imaging depth and large field of view Imaging depth and a large field of view are important because many biological samples are not single cell monolayers. Although its feasibility specialized has been demonstrated [79.Kim J. et al.Oblique-plane single-molecule localization microscopy for tissues and small intact animals.Nat. Methods. 2019; 16: 853-857Crossref PubMed Scopus (54) Google Scholar], imaging deeper than 10–50 μm in both SMLM and STED methods is challenging. The field would benefit from the use of engineered illumination, adaptive optics, image restoration algorithms, and multiphoton excitation. Ultimately, the best FN technology for live cell imaging must be selected based on the experimental setup and the precise biological question being addressed, and it is essential to include controls for possible phototoxic effects, such as conditions that are present that might cause damage to biological processes but are not considered. This should be done by using settings that cause less perturbation of biological conditions and can be achieved by troubleshooting the selection of appropriate labeling strategies and imaging conditions. For example, far-red light commonly used in the STED depletion laser is less phototoxic compared with the 405-laser used for SMLM at the same irradiance. The short answer to this question is 'no', because not all biological problems require molecular resolution to be solved. Careful consideration should be given to whether FN is not necessary for some biological questions. For example, FN is not required to determine whether a protein of interest (POI) is localized to lysosomes or mitochondria, but would be required to determine whether a mitochondrial POI is in the outer mitochondrial membrane or the inner mitochondrial matrix, which are ~20 nm apart. In general, the strength of FN is that it provides increased precision in the localization of individual biomolecules compared with diffraction-limited imaging (Figure 1B,C). A simple rule of thumb that can be used to decide whether FN is needed is to understand whether spatial information in the order of tens of nanometers allows the formulation of fundamentally different biological hypotheses for the process under investigation. At the same time, serendipitous observations were made and, in some cases, FN revealed new structures that were not observable at lower resolutions [14.Xu K. et al.Actin, spectrin, and associated proteins form a periodic cytoskeletal structure in axons.Science. 2013; 339: 452-456Crossref PubMed Scopus (888) Google Scholar]. For this reason, exploratory experiments should be considered. Very high-resolution imaging is informative, but aspects that could be ignored in conventional fluorescence microscopy become challenges in FN. Live FN would be the ultimate goal in biological studies, but it is still difficult to achieve due to the high phototoxicity of several FN technologies (Box 1). The field of FN is in its early stage and it is foreseeable that relevant advances will be made in the near future. At the same time, FN approaches in fixed samples are already changing the biology field; therefore, we concentrate here on fixed specimens (Figure 1D–F). In an experimental workflow for 'non-live' FN, the sample must first be fixed and the sample preparation must be optimized to avoid artifacts. After fixation, the FN approach requires the use of affinity-based labels, such as antibodies, to reveal molecular identities and position the reporter fluorophores in their proximity. To obtain relative or even absolute biologically relevant numbers from FN images (quantitative FN), further measures and considerations must be taken. In Box 2, we review the key issues to be considered when performing quantitative imaging at the nanoscale and explain why labeling density, imaging tool stoichiometry, and linkage error are key aspects in this context. Finally, in Box 3, we explain why there is not a single fluorophore that could perform best in all conditions.Box 2Quantitative FN: absolute versus observed number of moleculesQuantitative FN requires that the number of detected fluorophores matches (or, more precisely, correlates) with the real number of target molecules in the sample. Therefore, prerequisites for quantitative FN are a high and controlled labeling efficiency and the ability to detect all fluorophores.A high and controlled labeling efficiency is best achieved with monovalent affinity probes carrying a single (or at least a fixed number of) fluorophore reporting it (see Figure 2A in main text). Moreover, probes that utilize covalently linked labeling strategies that ensure that the targets are stably labeled with a single reporter should be preferred. The use of multivalent polyclonal reagents stochastically labeled with fluorophores (e.g., secondary antibodies carrying approximately one to six fluorophores) should be avoided since the correlation between the number of reporters and the target molecules might be inconsistent.Even in the ideal case in which all target molecules are decorated with an affinity-based label, the possibility exists that only some of the fluorophores decorating the affinity-based label are functional. Indeed, fluorophores might be inactivated, damaged, or not detected during the imaging procedure (see Figure 1E in the text). Detecting all single fluorophores in densely labeled samples is challenging, a problem known as fluorophore crowding. When the distances between fluorophores are on a single-digit nanometer scale, photophysical interactions occur, resulting in undesired alterations of fluorescence properties. For example, this happens between two fluorophores on the same structure, either by Förster resonance energy transfer (FRET) or even H-dimer formation if the two fluorophores are separated by molecular-scale distances [80.Ogawa M. et al.H-Type dimer formation of fluorophores: a mechanism for activatable, in vivo optical molecular imaging.ACS Chem. Biol. 2009; 4: 535-546Crossref PubMed Scopus (153) Google Scholar]. Notably, interactions between fluorophores have been reported for fluorophores decorating an antibody [81.Helmerich D.A. et al.Multiple-labeled antibodies behave like single emitters in photoswitching buffer.ACS Nano. 2020; 14: 12629-12641Crossref Scopus (13) Google Scholar], with one fluorophore serving as a 'super emitter' while others remain in the dark state. Some technologies, such as expansion microscopy, allow fluorophore crowding to be reversed, by physically creating a distance between molecules. Other technologies, such as DNA-PAINT, deal with the problem differently by modulating the concentration of the imaging probe or using light-controllable fluorophores [82.Raymo F.M. Photoactivatable synthetic dyes for fluorescence imaging at the nanoscale.J. Phys. Chem. Lett. 2012; 3: 2379-2385Crossref Scopus (60) Google Scholar] (see Figure 2D in main text).While relative quantification is more easily achieved, absolute molecule-counting approaches have been proposed for several approaches and a comprehensive review has recently been published on the quantification challenge [83.Hugelier S. et al.Quantitative single-molecule localization microscopy.Annu. Rev. Biophys. 2023; 52: 139-160Crossref Scopus (1) Google Scholar]. Importantly, all these methods need calibration to be benchmarked against known markers or biochemically (e.g., by quantitative western blot, liquid chromatography, or mass spectrometry).Box 3Fluorophore selection and why there are no one-size-fits-all solutionsA variety of fluorophores have been developed to fulfill the specific requirement of each FN technique (Table I). Factors that need to be considered when selecting a fluorophore include its chemical structure and charge, fluorescence quantum yield, and photostability (see Figure 2C in main text). While STED and expansion microscopy requires photostable fluorophores, SMLM and MINFLUX rely on molecules that reversibly switch between non-emitting and emitting states. For the possible switching mechanisms, we refer the reader to Figure 2D in main text.The most used fluorophore scaffolds in FN are cyanines and rhodamines. Among cyanines, Alexa Fluor 647 is considered the gold standard in SMLM and blinks in the presence of reducing agents and UV light [84.Berlier J.E. et al.Quantitative comparison of long-wavelength Alexa Fluor dyes to Cy Dyes: fluorescence of the dyes and their bioconjugates.J. Histochem. Cytochem. 2003; 51: 1699-1712Crossref PubMed Scopus (229) Google Scholar]. Rhodamines can relatively easily be modified to tune their spectral properties [85.Grimm J.B. et al.A general method to optimize and functionalize red-shifted rhodamine dyes.Nat. Methods. 2020; 17: 815-821Crossref PubMed Scopus (85) Google Scholar], membrane permeability [86.Lukinavičius G. et al.A near-infrared fluorophore for live-cell super-resolution microscopy of cellular proteins.Nat. Chem. 2013; 5: 132-139Crossref PubMed Scopus (648) Google Scholar], and equilibrium between the open fluorescent and the closed non-fluorescent forms. The regulation of this latter equilibrium induces live cell-compatible spontaneous blinking [86.Lukinavičius G. et al.A near-infrared fluorophore for live-cell super-resolution microscopy of cellular proteins.Nat. Chem. 2013; 5: 132-139Crossref PubMed Scopus (648) Google Scholar].Other frequently used fluorophores are based on coumarin, oxazine, or BODIPY scaffolds (see Figure 2E in main text). Coumarins are among the smallest fluorescent dyes and can be modified to generate variants with a large Stokes shift, which is advantageous for low background and multiplexing FN imaging [87.Nizamov S. et al.Phosphorylated 3-heteroarylcoumarins and their use in fluorescence microscopy and nanoscopy.Chem. Eur. J. 2012; 18: 16339-16348Crossref Scopus (45) Google Scholar]. Oxazines can be live cell compatible [88.Wombacher R. et al.Live-cell super-resolution imaging with trimethoprim conjugates.Nat. Methods. 2010; 7: 717-719Crossref PubMed Scopus (282) Google Scholar], have a red-shifted absorbance, high extinction coefficient, and the ability to 'blink' in buffers containing reducing oxidizing agents. Finally, BODIPY dyes are valued for their sharp absorption and fluorescence spectra combined with very high quantum yield and extinction coefficient [89.Kowada T. et al.BODIPY-based probes for the fluorescence imaging of biomolecules in living cells.Chem. Soc. Rev. 2015; 44: 4953-4972Crossref PubMed Google Scholar]. Although their application to FN is limited due to their highly hydrophobic nature and poor off-switching properties, recent low light-dose photoactivatable variants make them attractive for the field [90.Wijesooriya C.S. et al.A photoactivatable BODIPY probe for localization-based super-resolution cellular imaging.Angew. Chem. Int. Ed. 2018; 57 (12685–1268)Crossref Scopus (74) Google Scholar].In practice, fluorophores should be selected only after the most appropriate FN technique has been identified, always considering the specifications of the available instrument (e.g., lasers and detectors). The choice should also be driven by specific experimental needs, such as multicolor or live FN, the presence of autofluorescence in the sample, the need to reduce background, or counting molecules. An important consideration is that naked fluorophores may themselves have specific affinities for some cellular structures (e.g., lipophilic fluorophores may stain membranes). This should be evaluated when designing experiments, for example, by using fluorophores (reporters) without their targeting moiety as controls whenever necessary (see Figure 2B in main text). In general, we recommend that inexperienced FN users consult with their local expert to select the best dye for their application.Table IProperties of commonly used fluorescent dyesFluorophore classCoumarinsRhodaminesCyaninesBODIPYsOxazinesCommercial examplesAlexa Fluor 350, Pacific BlueAlexa Fluor 488, silicon-rhodamine, TMRAlexa Fluor 647, Alexa Fluor 555, Cy5BODIPY FL, BODIPY TMRAtto 655, Atto 680Spectral range (nm)360–700500–750500–1000500–700600–750Extinction coefficient (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 with FN methodsSTED, SMLMSTED, SMLM, MINFLUX, expansion microscopySTED, SMLM, MINFLUX, expansion microscopySTED, SMLMSMLM Open table in a new tab Quantitative FN requires that the number of detected fluorophores matches (or, more precisely, correlates) with the real number of target molecules in the sample. Therefore, prerequisites for quantitative FN are a high and controlled labeling efficiency and the ability to detect all fluorophores. A high and controlled labeling efficiency is best achieved with monovalent affinity probes carrying a single (or at least a fixed number of) fluorophore reporting it (see Figure 2A in main text). Moreover, probes that utilize covalently linked labeling strategies that ensure that the targets are stably labeled with a single reporter should be preferred. The use of multivalent polyclonal reagents stochastically labeled with fluorophores (e.g., secondary antibodies carrying approximately one to six fluorophores) should be avoided since the correlation between the number of reporters and the target molecules might be inconsistent. Even in the ideal case in which all target molecules are decorated with an affinity-based label, the possibility exists that only some of the fluorophores decorating the affinity-based label are functional. Indeed, fluorophores might be inactivated, damaged, or not detected during the imaging procedure (see Figure 1E in the text). Detecting all sin
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