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[Linawati Sutrisno](https://orcid.org/0000-0003-3085-9660), [Kewei Sun](https://orcid.org/0000-0002-1835-243X), [Katsuhiko Ariga](https://orcid.org/0000-0002-2445-2955)

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[Vision of Life’s Code: Molecular Probe Nanoarchitectonics for Deep RNA/DNA Illumination](https://mdr.nims.go.jp/datasets/d7dfe003-1eb5-4f2b-bcf3-8f68bb4e6ca9)

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Vision of Life’s Code: Molecular Probe Nanoarchitectonics for Deep RNA/DNA IlluminationVision of Life’s Code: Molecular Probe Nanoarchitectonics for DeepRNA/DNA IlluminationLinawati Sutrisno,* Kewei Sun, and Katsuhiko Ariga*Cite This: ACS Nano 2026, 20, 6493−6514 Read OnlineACCESS Metrics & More Article RecommendationsABSTRACT: Visualization of RNA and DNA provides a windowof opportunity for the early detection of multiple diseases andapproaches to manipulate them before any pathological processesoccur. However, current imaging systems often fail to meet thesedemands due to the need for multiple RNA and DNA chemicalprobes, which increases experimental complexity, reduces aware-ness of false positives, and limits imaging accuracy in complexbiosystems. In this work, we identify the key challenges associatedwith using multiple probes for RNA and DNA imaging and analyzethe mechanisms and performance of existing imaging tools. Thisarticle also introduces a conceptual framework and proposes amultidisciplinary framework to guide the development of next-generation imaging technologies. Furthermore, we highlight how nanoarchitectonics-based molecular design can enable single-stepmultiplexed RNA−DNA imaging. Our goal is to provide valuable resources for both biologists and probe developers for choosingsuitable molecular probes and further advancing their design, from initial concepts to commercial products�all from a biologist’sperspective.KEYWORDS: RNA, DNA, bioimaging, chemical probes, multiplex, in vitro, detection, biologist, chemist■ PROLOGUEGeneral Background: Journey to Real Use of NANOHuman civilization has developed alongside usable functionalmaterials, tools, and systems made from them. As societybecomes more globalized, scientific discoveries and engineer-ing inventions have the power to transform the quality of lifefor all of humanity. This became particularly evident during theIndustrial Revolution and the subsequent development ofscience and technology over the past century. Advances inproducing materials that perform specific functions and inprocessing those materials into precise structures that utilizetheir functions will create a more prosperous society.1,2 Thisperspective is being applied to the development of materialsthat address challenges in energy,3−6 the environment,7−10 andmedicine.11−14Two major developments have contributed to the advance-ment of functional materials since the 20th century. The first isthe advancement of various chemical fields. The developmentand systematization of organic chemistry,15−17 inorganicchemistry,18−20 polymer chemistry,21−23 coordination chem-istry,24−26 supramolecular chemistry,27−29 materials chemis-try,30−32 and biochemistry33−35 have created methodologiesfor the systematic production of many substances. The otherdevelopment is nanotechnology, which is supported by physicsand related technologies. Following the development ofmaterials chemistry, nanotechnology emerged in the mid-20th century and has advanced significantly since then.36,37Nanotechnology enables the observation38−40 and manipu-lation41−43 of structures at the atomic and molecular levels,revealing new phenomena in the nanoscale region.44−46 Thisprogress has significantly improved our scientific under-standing of nanoscale regions. Consequently, nanotechnologyhas become an attractive area of research.The goal of science and technology is to contribute tohuman and societal development. Translating the scientificknowledge of the nanoregion described above into usefulmaterials is essential.47−49 Historically, we must promote aconcept that integrates advances in materials chemistry withnanotechnology. One promising candidate is nanoarchitec-tonics, a postnanotechnology concept.50 It integrates materialschemistry and nanotechnology to construct functionalmaterials from nanounits, such as atoms, molecules, andnanomaterials.51,52 This methodology is highly general andReceived: December 20, 2025Revised: February 9, 2026Accepted: February 10, 2026Published: February 19, 2026Reviewwww.acsnano.org© 2026 The Authors. Published byAmerican Chemical Society6493https://doi.org/10.1021/acsnano.5c22282ACS Nano 2026, 20, 6493−6514This article is licensed under CC-BY 4.0Downloaded via NATL INST FOR MATLS SCIENCE (NIMS) on March 3, 2026 at 21:55:34 (UTC).See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.https://pubs.acs.org/action/doSearch?field1=Contrib&text1="Linawati+Sutrisno"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdfhttps://pubs.acs.org/action/doSearch?field1=Contrib&text1="Kewei+Sun"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdfhttps://pubs.acs.org/action/doSearch?field1=Contrib&text1="Katsuhiko+Ariga"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdfhttps://pubs.acs.org/action/showCitFormats?doi=10.1021/acsnano.5c22282&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsnano.5c22282?ref=pdfhttps://pubs.acs.org/doi/10.1021/acsnano.5c22282?goto=articleMetrics&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsnano.5c22282?goto=recommendations&?ref=pdfhttps://pubs.acs.org/doi/10.1021/acsnano.5c22282?fig=tgr1&ref=pdfhttps://pubs.acs.org/toc/ancac3/20/8?ref=pdfhttps://pubs.acs.org/toc/ancac3/20/8?ref=pdfhttps://pubs.acs.org/toc/ancac3/20/8?ref=pdfhttps://pubs.acs.org/toc/ancac3/20/8?ref=pdfwww.acsnano.org?ref=pdfhttps://pubs.acs.org?ref=pdfhttps://pubs.acs.org?ref=pdfhttps://doi.org/10.1021/acsnano.5c22282?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://www.acsnano.org?ref=pdfhttps://www.acsnano.org?ref=pdfhttps://creativecommons.org/licenses/by/4.0/independent of the type of material or its application.53,54Consequently, the concepts of materials nanoarchitecton-ics55,56 and molecular nanoarchitectonics57,58 have emerged.It is important that these technologies lead to thedevelopment of human society and improvements in theconvenience of daily life. This concept is deeply rooted amongscientists. Although the concept of nanoarchitectonics is notexplicitly stated, it is widely known that controllingnanostructures is necessary for a variety of applications.However, it cannot be said that research demonstratingfunctional improvements is scarce; for example, researchdiscussing the usefulness from the user’s perspective may belacking. Conversely, addressing these issues may clarify theimportance of molecular nanoarchitectonics.The main goal of this Review is to develop practicalmolecular nanoarchitectonics. As a target example, we wouldlike to focus on biological applications, specifically simulta-neous techniques for RNA/DNA imaging. Remarkableadvances have been made in bioscience and biotechnology inthe field of basic sciences. Medical technologies based on theseadvances have also made significant progress. In many cases,nucleic acids, such as DNA and RNA, are fundamental to thesebiological phenomena and technologies.59−61 While thesetargets are fundamental, it is interesting to consider whetheruseful technologies have been developed from the user’sperspective. Have molecular materials and technologies beendeveloped that can simultaneously distinguish between DNAand RNA, image them precisely in real time, and closelymonitor their behaviors? If not, what further developments areneeded? What contributions can molecular nanoarchitectonicsoffer? These are the questions addressed in this review. Thedetailed objectives and methodology are described in thefollowing sections.Focused Target: Identifying Critical Gaps in IntracellularRNA/DNA ImagingPrecise RNA/DNA imaging of complex biological systemsrequires a solid biological background for precise analysis, highrequirements for molecular probes, and accurate bioimagingsystems. However, in reality, most imaging systems fail to meetbiological research demands due to the need for multiple RNAand DNA probes, which might increase complexity, lowaccuracy in biological systems, and a lack of awareness of falsepositives. Such problems lead to time-consuming experimentsor complex analyses to collect reliable results, eventuallyleaving many RNA/DNA-related biological phenomenaunnoticed for decades. Unlike conventional review articles,which are intended only to collect all existing references onpopular topics, this review provides an overview of theuntapped areas in the bioimaging field, a list of currentlyoverlooked intracellular imaging problems, and an analysisfrom a biological perspective, inspiring researchers fromdifferent fields to explore new strategies relevant to theirquestions of interest.This review is also intended for general readers and has thefollowing objectives: (i) draw attention to long-standing, yetoften ignored problems in applying multiple RNA and DNAprobes for cellular imaging, (ii) provide an overview andcomparison of current techniques for simultaneous RNA andDNA imaging, (iii) examine their underlying mechanisms toinform strategies for designing next-generation imaging tools,(iv) highlight our recent discovery that might have thepotential to solve current problems, and (v) discuss newconcepts and provide guidelines along with their limitationsand possibilities to improve the development of RNA/DNAprobes for in-depth biological studies.Previous Reviews on RNA and DNA Imaging ProbesAlthough many reviews have explored strategies for designingRNA or DNA probes,62−66 most have been written from themaker’s perspective (chemists) rather than the user’s(biologists), which has caused many synthesized probes tofail to reach practical use. In this section, we present relevantrepresentative examples of existing reviews and identify severalimportant points that have been consistently overlooked inprevious reviews and current studies, which distinguish ourreview from others.The review article by Baghdasaryan and Dai discussed theNIR-II photoluminescence properties of gold molecularclusters for preclinical in vivo NIR-II imaging.62 The reviewsummarized the importance of NIR wavelengths for differenttypes of organs, including the vasculature, brain, kidney, liver,and gastrointestinal organs, as well as for molecularly targetedtumor imaging and theranostic treatments. It also discussed thedesign, synthesis, and basic mechanisms of current NIR-IIprobes for targeting specific biological targets. The review alsopointed out the need to consider controllable pharmacoki-netics and the biodistribution of clusters to avoid long-termtoxicity and highlighted several nanoparticles that havesuccessfully reached preclinical and clinical studies, such asgold nanoclusters (Au NCs). Although gold nanoclusters havea high potential for biomedical imaging, they still face a majorchallenge in the synthesis with high yield. Unlike larger goldnanoparticles, whose properties are size-dependent but notatomically defined, Au NCs require precise atomic-levelcontrol to ensure a uniform electronic structure, opticalproperties, and stability. One important point that this reviewand much of the current research often overlook is that NIRimaging is widely used for in vivo studies but is still rarelyapplied in in vitro studies.The review article by Le, Ahmed, and Yeo systematicallysummarizes recent advances in RNA imaging in both fixed andlive cells.63 These advances include fluorescence in situhybridization (FISH), single-molecule FISH (smFISH), roll-ing-circle amplification (RCA)-FISH, MERFISH, seqFISH, insitu sequencing (ISS), hybridization-based ISS, geneticallyencoded probes, such as CRISPR-associated (Cas) proteins forDNA targeting, the combination of fluorescently tagged RNApolymerase II with MS2 labeling of nascent mRNA to measureelongation rates, and multiple chemically synthesized probesfor imaging endogenous RNA in living systems, such as 2′-O-methyl ribonucleotides and phosphorothioate backbones. Thereview also highlighted several biological insights thatbiologists can gain through RNA imaging, including anunderstanding of RNA throughout its functional life cycle,such as transcription, splicing, localization, translation, anddegradation. Last but not least, the review also emphasized theneed to integrate high-throughput methods with large-scaleRBP−RNA interaction mapping approaches to capture themultidimensionality of RNA processing. Although this reviewfocuses on the importance of RNA imaging, it overlooks thesignificance of simultaneous RNA and DNA imaging, which isessential for achieving more accurate imaging and gainingdeeper biological insights.The review article by Dong and co-workers highlighted thecritical role of intracellular biomarker analysis for accurateACS Nano www.acsnano.org Reviewhttps://doi.org/10.1021/acsnano.5c22282ACS Nano 2026, 20, 6493−65146494www.acsnano.org?ref=pdfhttps://doi.org/10.1021/acsnano.5c22282?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-asdisease diagnosis.64 The review summarizes recent advances inDNA biosensors that utilize programmable DNA sequences asmolecular probes to monitor biomarkers. It outlined thefundamental structural components of DNA biosensors andtheir signal output mechanisms and discussed strategies forcellular internalization, including coincubation, nanocarrier-based delivery, and nanoelectroporation. The review alsocategorized the recent usage of DNA biosensors for detectingsmall molecules, RNAs, and proteins, as well as deliverymethods. In addition, the review noted that live-cell analysisoffers the advantage of enabling the long-term tracking ofspecific biomarkers over time. However, current DNAbiosensors face significant limitations due to their susceptibilityto degradation by intracellular nucleases, which often restrictthem to single-use detection events, which causes difficulty insustained intracellular monitoring. The review suggested thataddressing this limitation through the development ofinnovative delivery strategies could enable the nondestructivedelivery of DNA biosensors, thereby paving the way forcontinuous biomarker monitoring.To date, most reviews have focused on DNA or RNAimaging, overlooking the need for RNA/DNA multipleximaging, as well as the need for simple methods that areaccessible to a wide range of researchers, including non-specialists in the biological field.62−65 To our knowledge, noexisting review has identified the importance of simultaneousRNA/DNA imaging for in vitro studies or offered a systematicanalysis of the technologies capable of achieving it. This gapdistinguishes our review from others. Beyond summarizingexisting imaging tools, we also identify the conceptual barriersfor the first time and introduce methodologies that may helpovercome current RNA/DNA imaging limitations andaccelerate the development of next-generation chemical probesin a more precise manner, aligned with the needs of real-worldbiological research. Our goal is to help biologists select themost suitable approaches for specific biological research, andactivate future probe developers to design the next generationof RNA/DNA probes from conceptual ideas to commercialproducts from the biologists’ viewpoints.Figure 1. Building simplicity in complexity for high RNA/DNA imaging accuracy.ACS Nano www.acsnano.org Reviewhttps://doi.org/10.1021/acsnano.5c22282ACS Nano 2026, 20, 6493−65146495https://pubs.acs.org/doi/10.1021/acsnano.5c22282?fig=fig1&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsnano.5c22282?fig=fig1&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsnano.5c22282?fig=fig1&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsnano.5c22282?fig=fig1&ref=pdfwww.acsnano.org?ref=pdfhttps://doi.org/10.1021/acsnano.5c22282?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-as■ PROBLEMS, PROPERTIES, AND PROGRESSESLeap from Multistep to Single-Step Staining in a ComplexBiosystemMultiplex RNA/DNA imaging facilitates a powerful approachthat opens up new avenues for studying the structural andfunctional dynamics of cellular activity. This method not onlyanswers fundamental questions, such as “How do nucleus,nucleolus, and cytoplasm look in the cell?” and “How doesRNA or DNA look in various cellular activities?”. Moreimportantly, it is able to provide in-depth insights into RNAand DNA distribution as a result of the cell’s response to anybiological processes. Simultaneous visualization of RNA andDNA allows researchers to study how RNA interacts withchromatin organization in the nucleus, shedding light on themolecular processes involved in cell growth, differentiation,and even response to external and internal stimuli at the single-cell level.Combining DNA and RNA imaging probes within the sameexperiment is inherently problematic (Figure 1) because eachprobe is tailored to different targets and requires differentexperimental conditions.65−67 DNA is typically double-stranded and tightly packed within the nucleus or mitochon-dria, although it may be distributed to other cellular regionsand become single-stranded during certain biological pro-cesses. In contrast, RNA is predominantly single-stranded andstructurally more flexible. Such intrinsic differences influencehow DNA and RNA respond to chemical reactions; therefore,simultaneous RNA/DNA visualization under a single set ofconditions without RNA/DNA perturbation is required. Incomplex biosystems, these challenges become particularlypronounced. Overlapping signals due to structural andchemical similarities between DNA and RNA, probe−probeinteractions, and variable labeling efficiency can distort imagingresults. As a result, distinguishing real RNA or DNA signalsfrom artifacts often requires multiple controls and validations.Rather than providing deeper insights, combining separateDNA and RNA imaging protocols results in complicatedanalyses and reduces data reliability.Considering the facts presented above, it is urgent todevelop multiplex RNA−DNA cellular imaging that worksunder conditions compatible with both RNA and DNA in asingle step. Such approaches have several advantages. First,they enable the direct observation of spatial and functionalRNA/DNA relationships, providing a clearer and deeperunderstanding of how these interactions are regulated in realtime, with minimal processing time, less variability, and fewercontrol groups. Second, multiplex imaging allows thesimultaneous analysis of chromatin organization withinindividual cells, capturing dynamic molecular events that areoften lost in bulk or sequential measurements whileminimizing signal overlap from two different fluorophores.Third, by integrating both DNA and RNA information,researchers can better understand how structural genomefeatures influence transcriptional heterogeneity across differentcell types or states with minimal artifacts.Figure 2. Overview of biomedical imaging technologies and the focus of this review.ACS Nano www.acsnano.org Reviewhttps://doi.org/10.1021/acsnano.5c22282ACS Nano 2026, 20, 6493−65146496https://pubs.acs.org/doi/10.1021/acsnano.5c22282?fig=fig2&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsnano.5c22282?fig=fig2&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsnano.5c22282?fig=fig2&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsnano.5c22282?fig=fig2&ref=pdfwww.acsnano.org?ref=pdfhttps://doi.org/10.1021/acsnano.5c22282?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-asHistorical Flow: Transitioning from Difficult to Easy, yetInformation-Rich RNA/DNA Imaging TechniquesOver the years, the field of nucleic acid imaging has evolvedfrom early conventional approaches to more advancedtechniques capable of simultaneously visualizing RNA andDNA (Figures 2 and 3a).66,68−70 To address phototoxicity anddetect various RNA or DNA molecular conformations, Raman-based label-free methods for RNA and DNA have also beendeveloped. However, they still suffer from overlappingvibrational spectra, the need for linear unmixing, as well aslow resolution caused by weak signal intensity, which in turnleads to slow acquisition speeds during long-term imaging.71FISH, while capable of codetecting multiple RNA or DNAtargets that reach the genomic level,72,73 still suffers from theneed for multiple rounds of hybridization, imaging, andalignment. The weak signal strength of FISH often necessitatessignal-enhancement techniques,74 but these typically rely onhigh laser power or nonbiological window wavelength, thuscausing phototoxic effects during prolonged imaging.75Importantly, these complex methods require biologicalexpertise and are therefore not suitable for researchers whoare not specialists in the field.76 Overall, fluorescence imagingFigure 3. Overview of RNA/DNA imaging technologies. (a) Time sequence highlighting recent progress in simultaneous RNA/DNA imaging. (b)Comparative analysis of the existing methods for simultaneous RNA/DNA detection.ACS Nano www.acsnano.org Reviewhttps://doi.org/10.1021/acsnano.5c22282ACS Nano 2026, 20, 6493−65146497https://pubs.acs.org/doi/10.1021/acsnano.5c22282?fig=fig3&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsnano.5c22282?fig=fig3&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsnano.5c22282?fig=fig3&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsnano.5c22282?fig=fig3&ref=pdfwww.acsnano.org?ref=pdfhttps://doi.org/10.1021/acsnano.5c22282?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-aswith a chemical probe is the simplest approach for labelingRNA and DNA and can be performed without specializedbiological expertise. Although its high sensitivity allowsresearchers to easily detect changes in RNA or DNA withinbiological systems, both the molecular design and stainingprotocol must be well-optimized to obtain true-positiveimaging results (Figure 3b).Few Enough to Count on Our Fingers: Chemical Probes forSimultaneous Intracellular RNA/DNA DetectionGenerally, chemical probes recognize and interact with DNAand RNA through physical and chemical interactions, enablingthe specific visualization of nucleic acids in biologicalsystems.77 Intercalating probes insert planar aromatic struc-tures between stacked base pairs, stabilizing the nucleic acid−probe complex via π−π stacking. Groove-binding probesoccupy the major or minor grooves of DNA, forming hydrogenbonds and van der Waals contacts, often providing sequence orstructure selectivity. Many probes bind via electrostaticinteractions with the negatively charged phosphate backbone,allowing nonspecific associations that facilitate imaging incertain contexts. Sequence-specific recognition is achieved byprobes designed to form complementary base-pairing inter-actions with their targets, such as molecular beacons or peptideFigure 4. Selected examples of existing chemical probes for simultaneous RNA and DNA visualization. (a) Chemical structures of the existingcompounds plotted according to their excitation wavelengths. (b) Comparison of current compounds for dual RNA/DNA imaging. The dotsrepresent the peak excitation or emission wavelengths.ACS Nano www.acsnano.org Reviewhttps://doi.org/10.1021/acsnano.5c22282ACS Nano 2026, 20, 6493−65146498https://pubs.acs.org/doi/10.1021/acsnano.5c22282?fig=fig4&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsnano.5c22282?fig=fig4&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsnano.5c22282?fig=fig4&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsnano.5c22282?fig=fig4&ref=pdfwww.acsnano.org?ref=pdfhttps://doi.org/10.1021/acsnano.5c22282?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-asnucleic acids. Together, these mechanisms provide sensitiveand reliable visualization of DNA and RNA, supporting thestudy of their localization, structural dynamics, and functionalroles in both fixed and living cells.Considerable progress has been made in the development ofchemical probes for RNA and DNA imaging (Figures 3b and4). However, it still faces complications due to imaging issues,such as low photostability, nonspecific binding, sensitivity toenvironmental factors (e.g., pH, ionic strength), and the mostimportant challenge of simultaneously distinguishing RNAfrom DNA due to their similar structure. In the followingsections, we provide recent advances in the development ofchemical probes available for binding RNA and DNA, alongwith their respective advantages and limitations for cellularimaging, with special attention given to our recent discoveryabout the importance of nitrogen-containing core structures.Early work largely relied on Acridine Orange (AO), whichbecame a widely used tool due to its ability to selectively stainnucleic acids during 1940−1950.78 Several comprehensivereviews have documented these advances and the developmentof its derivatives,79−81 highlighting the optimized protocol,82,83proposed possibility mechanisms of AO staining,84,85 and itsdiverse applications in bioimaging.83,86,87 AO consists of anacridine core formed by three fused aromatic rings with twodimethylamino side groups and can exist in multipleresonance-stabilized isomeric forms, including para-quinone,ortho-quinone, and an uncharged free-base configuration,enabling various interaction modes with biomolecules. Infixed and living cells, AO uptake and localization might beaffected by the dynamic equilibrium between protonated anddeprotonated species, which influences its intracellulardistribution, aggregation state, and binding behavior. Inbiological environments, AO may exist as monomers orhigher-order aggregates, with aggregation depending on thelocal concentrations in specific biological targets. In fixed cells,AO is proposed to bind predominantly to single-stranded RNAwithin the nucleolus and cytoplasm through externalassociation and aggregate formation, which may be attributedto the structural flexibility of RNA. These binding states arereflected in the fluorescence behavior of AO, as microscopicvisualization under 436 nm excitation reveals concentration-dependent emission changes corresponding to the monomericand aggregated forms. Despite their utility, AO and itsderivatives suffer from intercalation between base pairs,which causes an increase in the length of the DNA helix,distorts the steric molecular structure, and alters the spatialcharge distribution.88 Additionally, their pH sensitivity and therequirement for UV excitation often cause unreliability inimaging;89 therefore, prestaining conditions require carefuloptimization to achieve accurate color in imaging. Althoughseveral acridine derivatives have been synthesized to increasemembrane permeability and facilitate RNA or DNA bind-Figure 5. Structural, chemical, and fluorescence properties of Acridine Orange (AO). (a) Molecular structure and resonance forms of AO. Theacridine core consists of three fused aromatic rings with two dimethylamino side groups. Resonance occurs among several possible isomeric forms,including para-quinone (top left and right), ortho-quinone (bottom left), and uncharged (unprotonated) free-base (bottom right) configurations.(b) AO behavior in fixed and live cells. Schematic representation of AO uptake and localization mechanisms, illustrating the equilibrium betweenprotonated (cationic) and deprotonated (free base) species. AO monomers are shown as cyan bars, and AO aggregates appear as magenta bars. Infixed cells, AO binds to single-stranded RNA in the nucleolus and cytoplasm primarily through external aggregate association rather thanintercalation, following the classical model. (c) Fluorescence behavior and microscopic visualization at varying AO concentrations under excitationat 436 nm.ACS Nano www.acsnano.org Reviewhttps://doi.org/10.1021/acsnano.5c22282ACS Nano 2026, 20, 6493−65146499https://pubs.acs.org/doi/10.1021/acsnano.5c22282?fig=fig5&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsnano.5c22282?fig=fig5&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsnano.5c22282?fig=fig5&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsnano.5c22282?fig=fig5&ref=pdfwww.acsnano.org?ref=pdfhttps://doi.org/10.1021/acsnano.5c22282?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-asing,90,91 they still suffer from biased visualization due to theiroversensitive properties (Figure 5a−c). Therefore, acridine andits derivatives are now more widely explored for theranosticsapplications rather than for long-term imaging (nowadayscommercialized).92To overcome the sensitivity problems of AO, cyanine-baseddyes, denoted as SYTO dyes, and their derivatives weredeveloped for RNA/DNA imaging (nowadays commercial-ized). Unlike AO, SYTO becomes highly fluorescent whenbound to nucleic acids.93,94 SYTO can label both RNA andDNA in live or dead eukaryotic cells and Gram-positivebacteria. SYTO derivatives have been explored and are nowcommercially available in blue, green, orange, and redfluorescent variants based on their excitation and emissionwavelengths. The SYTO family shares several features: abilityto cross nearly all types of cell membranes, including those ofbacteria and mammalian cells; high molar extinctioncoefficients greater than 50,000 cm−1 M−1 at their absorptionmaxima; low or almost no fluorescence when unbound (PLQYaround 0.01); and strong fluorescence when bound to nucleicacids (PLQY > 0.4, increases nearly 40-fold). Although theyshare similar features, each SYTO derivative differs inproperties such as cell permeability, the extent to which theirfluorescence increases when bound to nucleic acids, along withtheir excitation and emission profiles, DNA versus RNAselectivity, and overall binding affinity. Due to their high molarabsorption coefficients, they are compatible with a wide rangeof fluorescence-based instruments that use either laserexcitation or traditional broadband light sources such asmercury- or xenon-arc lamps. Although SYTO binds to bothRNA and DNA, its primary target and binding mechanism mayvary with cell type and biological environment. Interestingly, inthe use of nucleic acid dyes for staining for bacteria, RNA incertain species does not seem to bind SYTO 13 effectively.Despite their advantages, SYTO dyes exhibit overlappingDNA/RNA spectra, rapid photobleaching (faster thanHoechst 33342 and DRAQ5), limiting their suitability forprolonged or quantitative imaging that requires high-intensitylaser illumination.LUCS-9 represents a refined SYTO family member andexhibits fluorescence upon binding to nucleic acids, withemission peaks at around 500 nm for DNA and 504 nm forRNA. Such slight spectral differences and low photostabilityalso limit its capability for simultaneous imaging of bothnucleic acid types. However, its cell-permeant ability makes itFigure 6. Historical development of pyrazinacenes: from molecular design to their first exploration in bioimaging. (a) Electronic absorption spectraof pyrazinacenes with an increasing number of fused pyrazine rings. (b) RNA−DNA and multiple cell-state discrimination using a singlefluorophore (TEG8-N14) under NIR excitation.ACS Nano www.acsnano.org Reviewhttps://doi.org/10.1021/acsnano.5c22282ACS Nano 2026, 20, 6493−65146500https://pubs.acs.org/doi/10.1021/acsnano.5c22282?fig=fig6&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsnano.5c22282?fig=fig6&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsnano.5c22282?fig=fig6&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsnano.5c22282?fig=fig6&ref=pdfwww.acsnano.org?ref=pdfhttps://doi.org/10.1021/acsnano.5c22282?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-aswell-suited for both live and fixed-cell microscopy, as well asflow cytometry.To overcome the photostability problem of cyanine-baseddyes, Han and colleagues developed a highly photostable dye,cationic carbon quantum dots (CQDs), capable of distinguish-ing fluorescence upon binding to single-stranded RNA(ssRNA) and double-stranded DNA (dsDNA) in live cells.67The cationic charge was introduced to the CQDs through theaddition of polydopamine and PPh3+, enabling the nanodots tointeract with both dsDNA and ssRNA via multiple non-covalent interactions, including ionic, π−π, and hydrogenbonding. Different interactions have been proposed for dsDNAand ssRNA: the high structural rigidity of dsDNA confinesCQDs within the grooves, resulting in enhanced fluorescencefrom isolated particles, whereas flexible ssRNA acts as a CQDconcentrator, bringing multiple CQDs into close proximity.Several attractive features make these CQDs promising forbioimaging, including compatibility with STED microscopy forhigh-resolution imaging, high photostability (higher thanHoechst 33342), and cell permeability, which have beendemonstrated by their ability to monitor cell division and C.elegans growth. Despite their promising capabilities, therequirement for UV illumination restricts their applicationfor prolonged imaging at short time intervals.TO3-CN, a representative example of thiazole orangederivatives, was subsequently designed to improve thephotostability of the previous development.95 Specifically, theintroduction of a cyano (CN) group into the trimethine chainof the classical red-emitting TO-3 dye enhances photostability,fine-tunes spectral properties, and modulates interactions withnucleic acids.96 TO3-CN exhibited several attractive features,including a large fluorescence Stokes shift (>40 nm), highquantum yield (>0.7), and low cytotoxicity. It showedenhanced brightness upon binding to DNA (approximately7-fold increase, molar extinction coefficient more than 50,000cm−1 M−1) and RNA (around 7-fold increase). However, theclose spectral overlap of DNA and RNA limits its ability todiscriminate between the two simultaneously. Therefore, underthe same observation conditions (excitation 559 nm, emission575−620 nm), fluorescence in MCF-7 cells was observed inboth the nucleolus and nucleus.DR1, a cyanine-based dye, provides a better strategy thanTO3-CN for RNA/DNA spectral discrimination by bindingDNA through both the major and minor grooves and RNAthrough electrostatic interactions.97 Although the distinctspectral signatures of DNA and RNA allow simultaneousdiscrimination, their low photostability is primarily due to thedouble-bond structure, which makes them highly prone todegradation during prolonged imaging. Given its potentialphototoxicity, low photostability, and limitations for long-termimaging, DR1 is better suited for high-content screeningapplications rather than real-time imaging.To date, the majority of fluorescent probes used forintracellular imaging rely on UV−visible excitation, largelydue to the limited sensitivity of conventional fluorescencemicroscopy systems in the near-infrared region. As a result,intracellular probes that operate efficiently under NIRexcitation remain rare, and their successful implementationrepresents a significant technical breakthrough. In this context,we recently reported a significant advance that overcomescurrent probe limitations and microscopy constraints bydeveloping an optimized staining strategy for N-rich structuredcompounds (denoted as TEG8-N14) to simultaneouslyvisualize RNA and DNA with dual near-infrared excitation(Figure 6a,b).98 Such NIR excitation not only minimizesphototoxicity and spectral crosstalk but also shows broadapplicability for measuring multiple types of cell injury indifferent cell types. Docking simulations indicate that itsbinding to DNA is based on multiple interactions, starting withC−H···π interactions, groove binding, and anion−π inter-actions between the phosphate backbone and the electron-deficient extremities of its core unit. In contrast, for RNA, theinteractions are more variable and depend on its secondarystructure. One advantage of this probe is that it binds to DNAvia groove binding rather than intercalation, which causeslower cytotoxicity and avoids distortion of the DNA doublehelix. Groove binding also reduces interference with DNA-binding proteins compared to intercalation, allows for higherbinding selectivity when the staining protocol is properlyoptimized, and provides higher photostability compared tocommon intercalating dyes. Interestingly, its photostability iscomparable to that of SiR-DNA, one of the most photostableprobes developed to date. Due to its aggregation, the probeexhibits reduced cell permeability and low cellular brightnesswithout the addition of a proper surfactant. Therefore, futuredevelopment should focus on strategies to improve the cellpermeability and cellular brightness and modify the chemicalstructure.In summary, although there are many probes that are able tobind DNA and RNA in cells, to date, only four types ofprobes�AO and its derivatives, CQDs, DR1, TEG8-N14�have the capability for simultaneous DNA and RNA imaging.These four examples provide further possibilities as afundamental framework for designing chemical probes withproperties that extend beyond the simple combination ofindividual RNA or DNA probes. Specifically, such dual-function probes are essential for flow cytometric studies,99high-throughput screening,97 histochemical observation incells, bacteria, or viruses,100 observation of chromatinstructural changes during repair pathways,101 cell cycle,67 orstress,102 which are crucial for advancing diagnostics andtherapeutic developments.■ PROMISING PROBES AND PROCEDURESWhat Kind of Fluorophore is the Best from a Biologist’sPerspective?Although a perfect probe does not exist, critical designelements are essential for its optimal performance. In thefollowing sections, we outline several key design criteria fordeveloping chemical probes along with the limitations thatsignificantly hinder their performance. We discuss severalunusual concepts that have changed the traditional concept ofchemical probe development. We also propose a way to adjustthe specific application of probes, which is often overlooked bymost researchers in the field.Biological Window: Gateway for Universal ApplicationThe complex structure of biological cells and tissues oftenleads to opacity, primarily due to unwanted light scattering andabsorption in the biological environment, which limits thepenetration depth of optical imaging and causes unwantedautofluorescence from endogenous fluorophores.103−105 Inmany cells and tissues, the scattering coefficient exceeds theabsorption coefficient by 10- to 1000-fold, making scatteringthe main factor that restricts both imaging depth and spatialresolution in conventional microscopy (Figure 2). AlthoughACS Nano www.acsnano.org Reviewhttps://doi.org/10.1021/acsnano.5c22282ACS Nano 2026, 20, 6493−65146501www.acsnano.org?ref=pdfhttps://doi.org/10.1021/acsnano.5c22282?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-asvarious probes targeting nonbiological window regions havebeen developed,106 autofluorescence of specific cell types andphototoxicity during long-term imaging remain a concern,particularly when visualizing light-sensitive parts, such as DNAand RNA,107 which can potentially compromise the accuracyof the results. For cellular imaging applications, the focus ofthis review, the selection of an appropriate NIR biologicalwindow depends on the experimental context.4,108,109 Forexample, 3D cell cultures, such as spheroid, organoid, or in vivoimaging, require longer wavelengths for deep imagingcompared to 2D systems.110−113 In addition, different celltypes have different degrees of endogenous fluorophores,making it crucial to carefully choose fluorescence excitationand emission wavelengths tailored to each specific application.Photoluminescence Quantum Yield is Not the PrimaryConsiderationAlthough the photoluminescence quantum yield is animportant photophysical parameter, a very high quantumyield is not necessarily the most critical factor, as probes maybehave differently once localized to a specific area or interactwith specific biomolecules in biological media.114 Some probesmay show fluorescence enhancement after they interact withbiomolecules. Therefore, cellular brightness after probeinternalization should be given greater consideration.115Oversensitivity: A Source of Analytical ComplexityAlthough probe sensitivity is essential for sensing applica-tions,106,114,116 multiple sensitivities complicate data interpre-tation and increase the requirement for rigorous calibration.For example, AO is sensitive to pH, aggregation, andprotonation.117 Therefore, when detecting RNA changes incertain diseases with AO, many questions arise: “Are theobserved signals due to RNA changes, aggregation changes, orpH differences?” or “Are there any nonspecific binding thatcauses a difference in fluorescence intensity?” Such excessresponsive probes can amplify minor fluctuations, particularlyin sensing applications, potentially leading to misleadingfluorescence intensity quantification readouts unless carefullycontrolled and standardized. However, by using such probes,we can still accurately extract information about cell kinetics ormorphological features. In summary, probes with just specificsensitivity are much preferred over those with high sensitivity.Trade-Off between Brightness and PhotostabilityBy avoiding fast photobleaching, a probe can be used not onlyfor quantitative and long-term imaging but also in super-resolution microscopy,112,118 which provides more detailedinsights into biology-related processes. However, achieving abalance between brightness and photostability remains a majorchallenge in probe design, especially due to the aggregation-induced quenching effect or any unwanted interactionexhibited by the majority of molecular probes in biologicalmedia.63,119,120 Bright molecular probes often undergo rapidphotobleaching under continuous illumination. In contrast,highly photostable fluorophores may have low brightness,reducing their detectability.121 To maximize a probe’s utility inlong-term imaging and balance photostability and brightnessover time, careful molecular design must be optimized toachieve both properties effectively.122,123 A good example of amolecular design that balances photostability and cellularbrightness is pyrazinacene, which was developed by extendingthe number of fused pyrazine rings and introducing nitrogenatoms to enhance stability, as well as by TEGylation toimprove its water solubility.124 Incorporating nitrogen atomsinto an aromatic framework leads to tunable molecular orbitalFigure 7. Distinct morphological features from apoptosis to necrosis/necroptosis stages can be distinguished using RNA/DNA staining. Reprintedfrom ref 98. Copyright 2025, AAAS, licensed under CC BY 4.0.ACS Nano www.acsnano.org Reviewhttps://doi.org/10.1021/acsnano.5c22282ACS Nano 2026, 20, 6493−65146502https://pubs.acs.org/doi/10.1021/acsnano.5c22282?fig=fig7&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsnano.5c22282?fig=fig7&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsnano.5c22282?fig=fig7&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsnano.5c22282?fig=fig7&ref=pdfwww.acsnano.org?ref=pdfhttps://doi.org/10.1021/acsnano.5c22282?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-asenergies, resulting in an improved electron affinity andenhanced oxidative stability. For instance, pyridinic nitrogenatoms act as electron-withdrawing units, stabilizing azaar-omatic systems relative to their parent hydrocarbon analogues.In particular, this stabilization is associated with a lowering ofthe frontier molecular orbital energies and depends not only onthe number of nitrogen atoms introduced but also on theirprecise positions within the aromatic backbone. Thesepositional effects influence the oxidation behavior, suscepti-bility to electrophilic attack, binding affinity, chemicalinteractions, and local electron density in biological environ-ments, ultimately contributing to enhanced chemical stabil-ity.125 Although pyrazinacene has a self-quenching effect inaqueous media, its fluorescence “switch-on” properties uponinteraction with RNA or DNA facilitate bioimaging applica-tions while maintaining both photostability and brightness,allowing the monitoring of different types of cell injury (Figure5a).Cell Permeability: Not Always an Obstacle to BioimagingApplicationsCell permeability is a critical factor for tracking dynamicprocesses in living cells. Due to the highly specific nature ofnuclear pores, it remains difficult for small molecular probes orother organic molecules to pass through and enter the nucleus.Therefore, tailoring the structure of chemical probes or addinga cell-penetrating agent must be considered for imagingbiological events in the nucleus. However, the probe’s limitedpermeability does not necessarily preclude its practicalapplication. For example, propidium iodide and DAPI havebeen widely employed to detect plasma membrane rupture andother forms of cell death.125−128 Therefore, even if a probe isimpermeable, its specific application must be carefully tailoredbased on its properties. Additionally, such probes are stillsuitable for imaging fixed cells that require permeabilization. Inthe context of live-cell imaging, cell permeability is a primarycriterion, but any potential changes during tracking must alsobe taken into account to ensure accurate and reliable imagingquantification (Figure 6b).Multiplexing: Mapping Organelle Interactions withCompatibility Across ProbesMultiplexed fluorescence imaging enables the simultaneousdetection of multiple targets in a cell, revealing the relationshipbetween targets in a single step.114,129,130 Without thiscapability, it is difficult to identify relationships among signals,analyze their interactions, and understand how these biologicalprocesses malfunction under pathological conditions. As asimple example, consider a case where signal A is high in a cellwhile signal B is low, and vice versa, perhaps because Asuppresses B. Imaging A and B in separate cells would fail toreveal this relationship; only by measuring both signalssimultaneously in the same cell with the same probes canthis interaction be clearly observed.131 Another case,identifying changes in a single organelle, is not always sufficientto understand cell states. For example, in the late stage ofnecrosis, some cells become bright and smooth without RNAdisruption in the nucleolus, while others do not. Similarly,during the senescence stage, observing only DNA is insufficientfor the early detection of metabolic disruption (Figure 7).98Therefore, RNA and DNA signals should be detectedsimultaneously and, if possible, multiplex probes should beused without interference of their compatibility to betterunderstand the cell state in an easier and more precise way.Retention Time: A Barrier to Recording a CompleteCellular HistoryThe retention time of a fluorophore in a subcellular organelledirectly influences its long-term imaging performance. A probeshould remain localized to its target without disturbing cellularmetabolic activity while also avoiding nonspecific leakagecaused by serum or other extracellular components.132Fluorophore−target interactions can change over time due toprobe diffusion, weak binding affinity, target dynamics, orenvironmental changes. Such time-dependent specificity mustbe considered and evaluated when designing experiments,especially for live-cell or long-term imaging studies, to ensurethat the signal fidelity reflects the target rather than the artifact.Both inadequate retention and prolonged sequestration cancompromise signal specificity and biological relevance. ByFigure 8. Changes in RNA and DNA distribution serve as markers for identifying the specific phases of the cell cycle. Reprinted with permissionfrom ref 67. Copyright 2019, Wiley-VCH.ACS Nano www.acsnano.org Reviewhttps://doi.org/10.1021/acsnano.5c22282ACS Nano 2026, 20, 6493−65146503https://pubs.acs.org/doi/10.1021/acsnano.5c22282?fig=fig8&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsnano.5c22282?fig=fig8&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsnano.5c22282?fig=fig8&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsnano.5c22282?fig=fig8&ref=pdfwww.acsnano.org?ref=pdfhttps://doi.org/10.1021/acsnano.5c22282?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-asachieving these two important criteria, the complete cellularhistory can be recorded in a more precise manner (Figure 8).Interference of Other Biomolecules: Impact on ImagingAccuracyNonspecific binding to proteins, lipids, or other biomoleculesin the cell might lead to off-target fluorescence, quenching,local photothermal effect, or spectral distortion. Optimizingchemical probes to reduce such interactions (e.g., withzwitterionic) is crucial for achieving high target selectivity,particularly in quantitative imaging in a crowded bioenviron-ment.133,134Imaging Complexity Arising from Synergistic TherapeuticDiagnostic AttemptsMost studies focus on the synergistic use of imaging andtherapeutic functions to eliminate and image cancer cells.79,135However, there are several important considerations for such asynergistic approach, such as “What happens to probedetection once cancer cells begin to die? Will the probe stillprovide accurate quantitative imaging, or will it bind to otherbiomolecules due to changes in the biological environment?Moreover, how reliable is the quantitative sensing capabilityduring the dynamic treatment process?” Taken together, suchfunctional-purpose probes must be carefully designed to ensurea meaningful interpretation of imaging outcomes.Beyond Simple: Considerations for Accurate Selection andFuture DevelopmentIn this section, we outline several considerations that bothbiologists and probe developers should consider whendesigning bioimaging experiments.Complexity Limits In-Depth Understanding of HowStaining WorksAdvancing next-generation probes requires an in-depthunderstanding of how they interact with their biologicaltargets. Once inside the cells, probes encounter a highlycrowded environment composed of biomacromolecules, ions,membranes, and other cellular components. This complexitymakes it challenging to identify the precise binding sites anddetermine how these interactions influence imaging outcomes(Figure 9).136 A major barrier in understanding probe stainingmechanisms arises from the combination of multiple potentialchemical binding sites and the heterogeneous nature of thecellular environment. For instance, although TEG8-N14 is oneof the most promising probes for RNA/DNA imaging becauseof its high photostability and near-infrared absorption, itspractical application remains limited by its low scalability.Incorporation of nitrogen into the acene core can induce anumpolung of the electronic properties, thereby convertingacenes into a class of electron-transporting materials. However,when nitrogen atoms are arranged in the ortho or metapositions, the structural motifs exhibit increased susceptibilityto nucleophilic attack, which complicates their purification dueto facile deprotonation. Therefore, further development of itschemical design, such as structural modification andincorporation of cell-penetrating peptides, liposomes, or anytype of nanocarrier, is required to overcome the problemsmentioned above. Hence, a deeper understanding of thebinding mechanism is necessary for further optimization.Although docking simulation studies have suggested thepossibility of groove binding with DNA and hydrogen bondingwith RNA, factors such as multiple binding sites, protonation/Figure 9. Protonation, redox, aggregation, and solvatochromic responsiveness of the N14 framework. (a) Structure of the oxidized product (Ox) ofPh4H2N14HEPT. (b) Chemical structures of monoanion (MA) and dianion (DA). (c) Tautomers of Ph4H2N14HEPT (T0, T1, T2, and T3)accessible through concerted double-proton shifts (other possible tautomers are not considered here). Structures of the selected protonatedtautomers of Ph4H2N14HEPT calculated in this study. (d) Spectral changes associated with aggregation states. (e) Spectral variations observed insolvents with different polarities, demonstrating solvatochromic behavior.ACS Nano www.acsnano.org Reviewhttps://doi.org/10.1021/acsnano.5c22282ACS Nano 2026, 20, 6493−65146504https://pubs.acs.org/doi/10.1021/acsnano.5c22282?fig=fig9&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsnano.5c22282?fig=fig9&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsnano.5c22282?fig=fig9&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsnano.5c22282?fig=fig9&ref=pdfwww.acsnano.org?ref=pdfhttps://doi.org/10.1021/acsnano.5c22282?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-asdeprotonation states, and the type of DNA or RNA may alsoinfluence their performance. Therefore, a detailed under-standing of its mechanism is necessary before proceeding tofurther development.98,124 Although theoretical calculationsare widely used to investigate such mechanisms, they remaininsufficient for fully elucidating the staining mechanism.Owing to recent developments, artificial intelligence andmachine learning have contributed to the prediction ofbiological responses and customization of molecular recog-nition for guiding probe design and personalized drugdelivery.137 Despite these advances, the lack of high-qualityand standardized AI data sets often leads to fragmented data,inconsistent reports, and a lack of coherence between surfacechemistry and biological results. Such facts, in turn, make datainterpretation difficult and ultimately create hurdles forvalidation and reproducibility.One of the most reliable methods to reveal such a stainingmechanism is direct molecular observation and in situcharacterization of different chemical structures. As long-termdevelopment goals, the following two vital areas arerecommended:Scanning Tunneling Microscopy/Atomic Force Mi-croscopy (STM/AFM). Imaging DNA and RNA withfluorescent molecules at atomic resolution is extremelychallenging, but surface techniques such as STM/AFM mayoffer a possible path toward achieving this goal. STM/AFM areimportant tools in on-surface chemistry, especially for resolvingcarbon-based nanostructures synthesized on solid sub-strates.138−141 In earlier studies, STM/AFM enabled thecharacterization of DNA at the solid−liquid interface;however, the resolution was limited, making it difficult toachieve high resolution.142,143 Drying the deposited solutionallows the sample to be measured under ultrahigh vacuum,which helps improve the resolution.144,145 More advancedultrahigh-vacuum deposition techniques for macromoleculesare still needed. Fortunately, recent progress in electrospraymethods has allowed direct deposition onto metal substratesunder ultrahigh-vacuum conditions, enabling high-resolutionSTM/AFM imaging of species such as proteins,146 peptides,147glycans,148 and DNA molecules.149 Even glycans bonded toproteins and lipids can be observed and studied by STM at thesingle-molecule level.150 Similarly, DNA or RNA together withfluorescent molecules can be deposited onto a surface usingelectrospray techniques and subsequently investigated bySTM/AFM under ultrahigh-vacuum conditions. In addition,we propose an improved approach: first, DNA/RNA isdeposited onto the substrate by electrospray, and thenfluorescent molecules are deposited onto the partially DNA/RNA-covered surface using organic molecular beam epitaxy(OMBE), allowing them to bind directly to the DNA/RNA onFigure 10. Atomic-scale characterization of ssDNA and molecular dynamics simulations. (a) STM image of hydrated ssDNA after spray depositionat room temperature. The right side shows a representative ssDNA structure on Au (111) obtained from molecular dynamics (MD) simulations.(b) STM image of a dehydrated single 20-cytosine ssDNA oligomer after annealing at 440 K, and the corresponding high-resolution constant-height AFM image, both acquired with a CO-terminated tip. (c) STM image of self-assembled dehydrated ssDNA oligomers after heating to 500 K,and the corresponding AFM image. (d) MD simulation side and top views depicting the adsorption of a water droplet containing one ssDNAstrand onto Au(111) (water is shown as a transparent surface). (e) 500 ns MD simulation at 500 K capturing the diffusion-driven assembly of twostrands via intermolecular interactions. Reprinted from ref 149. Copyright 2019, Springer Nature; licensed under CC BY 4.0.ACS Nano www.acsnano.org Reviewhttps://doi.org/10.1021/acsnano.5c22282ACS Nano 2026, 20, 6493−65146505https://pubs.acs.org/doi/10.1021/acsnano.5c22282?fig=fig10&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsnano.5c22282?fig=fig10&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsnano.5c22282?fig=fig10&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsnano.5c22282?fig=fig10&ref=pdfwww.acsnano.org?ref=pdfhttps://doi.org/10.1021/acsnano.5c22282?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-asthe substrate. Next, the binding sites between DNA/RNA andfluorescent molecules can be visualized by using STM/AFMcharacterization. This information on the binding sites is veryimportant as it reveals how the interactions occur and the typesof interactions that take place. Moreover, scanning tunnelingspectroscopy (STS) can detect the highest occupied molecularorbital (HOMO) and the unoccupied molecular orbital(LUMO) of fluorescent molecules.151 When these moleculesbind to DNA/RNA, their electronic states shift, leading tovariations in the HOMO−LUMO gap, which indicates thebinding mode and interaction strength. STS enables the directprobing of these changes, offering a clearer picture of howfluorescent molecules interact with DNA/RNA and thestrength of these interactions. For example, utilizing a CO-terminated tip for NC-AFM enables the imaging of ssDNA andthe corresponding self-assembled structures with subnanom-eter resolution (Figure 10a). This high-resolution techniqueclearly reveals the folded conformation and carbon backbonestructure of dehydrated ssDNA oligomers on the Au(111)surface (Figure 10b,c). The acquired experimental imagesshow excellent agreement with the structures obtained fromthe molecular dynamics simulations (Figure 10d,e), confirmingthe observed structural details. This approach provides apowerful pathway for the atomic-scale characterization ofsingle biomolecular strands.In Situ Characterization. To better understand theunderlying staining mechanisms of molecular probes, it isnecessary to compare the different chemical structures of theprobes and evaluate their performance in biological environ-ments. Different chemical structures, such as alkyl chains,donor−acceptor motifs, and other functional groups, can affectthe binding modes, photophysical properties, cell permeability,and interactions with biomolecules. Therefore, in-depthinvestigations using techniques such as spectroscopic measure-ments, isothermal circular dichroism, microscale thermopho-resis, or X-ray crystallography are required to clarify thestructure−function relationships that influence probe selectiv-ity, brightness, and cellular behavior (Table 1). These insightshelp improve probe selectivity, brightness, and behavior in cellsand are essential for designing more reliable and effectiveprobes for imaging and other biological applications.Rethinking the Use of AI for Data Analysis with Attentionto Ethical ConcernsAnalyzing large and complex imaging data sets is time-consuming and requires high-level microscopy expertise. AI-based platforms, such as Leica’s Aivia and Imaris, have beendeveloped to automate segmentation and quantification.Table 1. Techniques Used to Study Probe−DNA Binding with Site-Specific Interactionstechnique example applicationcircular dichroism (CD)spectroscopymonitors conformational changes in the DNA structure and identification of groove binding or intercalationmicroscalethermophoresis (MST)measurement of direct binding affinity (Kd) of a molecule to DNA to reveal sequence preference, like preferential binding complexes forGC-rich vs AT-rich sequencesthermal melting (Tm)experimentsdetects changes in DNA duplex stability in the presence of a drug, such as the evaluation of sequence-specific thermal stabilizationX-ray crystallography enables atomic-level resolution of compound−DNA interactions, such as structural elucidation of binding modes like intercalation withsequence-specific preferences by complexes relative to groove bindingin-liquid AFM enables visualization of DNA topologies in hydrated form, such as detection of DNA triplex formation at base-pair resolutionFigure 11. Overlooked problems in fluorescence imaging and proposed solutions for improving its accuracy.ACS Nano www.acsnano.org Reviewhttps://doi.org/10.1021/acsnano.5c22282ACS Nano 2026, 20, 6493−65146506https://pubs.acs.org/doi/10.1021/acsnano.5c22282?fig=fig11&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsnano.5c22282?fig=fig11&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsnano.5c22282?fig=fig11&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsnano.5c22282?fig=fig11&ref=pdfwww.acsnano.org?ref=pdfhttps://doi.org/10.1021/acsnano.5c22282?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-asAlthough these platforms offer many benefits, they must beapplied with caution. Several limitations, such as insufficienttraining data, variable data quality, and algorithmic biases, canaffect the data reliability and raise ethical concerns. Establish-ing guidelines for the responsible use of AI in biologicalimaging and performing deep learning with professionals iscritical to ensure scientific validity, reproducibility, andtransparency.152,153Solid Biological Knowledge Is a Prerequisite for AccurateResultsBiological systems are highly dynamic and interconnected, andeven small differences in cellular states or environments cansignificantly affect experimental results. Accurate interpretationof imaging data, therefore, requires a deep understanding ofbiological principles and context. Without this foundation andhigh standardization, there is a risk of misattributing artifacts,overlooking context-dependent phenomena, or drawingambiguous conclusions, which can obscure the true underlyingprocesses. Therefore, standardized protocols are needed tominimize linkage errors.Careful Selection of Each Experimental Component forCharacterizationCharacterization is essential before advancing to the next stageof probe development. Below, we outline several importantconsiderations for probe characterization.Sensitivity of Detection Equipment. The performanceof each probe is determined by the characterization of itsphotophysical properties after binding to the cells. Keycomponents, such as detectors, lasers, objective numericalapertures, and other optical elements, must be properlycalibrated to maintain stable signals and reduce noise duringthe experiments. Inadequate calibration and the lack of apositive control can lead to false positives and mislead theinterpretation of the probe behavior (Figure 11).Fixation Selection and Why There Are No One-Size-Fits-All Solutions. At the initial stage of sample labeling,fixation should preserve the structural integrity of the specimenthat closely reflects the state of the living sample; however, thisprocess is susceptible to the introduction of artifacts. There isno universal fixation protocol, as a fixative that performs wellunder one set of conditions may be suboptimal underanother.65 For example, 4% paraformaldehyde (PFA) is widelyused because it provides good preservation of morphology;however, it penetrates samples relatively slowly. Penetrationcan be improved by the addition of 10% ethanol, although thismay alter organelle morphology. Cold methanol fixation iscommonly employed for certain cytoskeletal structures but failsto preserve cellular membranes. Glutaraldehyde offers superiorstructural preservation, yet it can modify epitopes, reduceimmunogenicity, and impair the performance of affinity-basedlabels. Membrane permeabilization, while frequently required,inevitably extracts lipids and disrupts cellular membranes andorganelles. To circumvent the need for permeabilization,smaller or membrane-permeant affinity-based probes can beemployed. Finally, incomplete fixation may leave somebiomolecules moving, leading to apparent protein localizationthat differs from their native state. Therefore, testing multiplefixation strategies is generally recommended for each targetstructure or model system.Cell Culture Platform. The most important considerationwhen choosing a cell culture platform for imaging is its abilityto closely mimic the native tissue environment. Factors such ascell type, extracellular matrix composition, and culture platformTable 2. Representative Application of Simultaneous Imaging of the Nucleoli, Cytoplasm, and Nucleusaprobeobservation condition(λex/λem, nm) model system application refsAO 457/525 (DNA) MO3 cell oligodendrocyte injury detection 87457/625−645 (RNA) MEF and NIH-3T3 cells apoptosis, necrosis, and necroptosis detectionThy1-YFP mouse optic nerve chemical ischemia detectionmouse spinal cord spinal cord injury detection488/530 (DNA)488/650(RNA)lens epithelial cell galactosemia detection 154488/530 (DNA)488/640(RNA)histopathological staining of primary renalcell carcinoma (RCC)primary renal cell carcinoma (RCC) detection 155467/520 (DNA) X-ray(radiotherapy)human musculoskeletal sarcoma photodynamic or radiotherapy for human musculoskeletalsarcomas156N.A. zebrafish oogenesis stage detection 157490/510−560 (DNA)490/600−650 (lysosomes)mixed glial cell culture (astrocyte/microglia)discriminatory marker of microglia in different types ofastrocytes158488/580−630 (RNA) duffy positive and duffy knockoutmiceperipheral blood of anemic miceidentification of infected erythrocytes and reticulocytes inmalariadetermine the maturity of reticulocytes159N.A. chemo-sensitive mouse osteosarcoma cell photodynamic therapy of multidrug resistance (MDR) ofmouse osteosarcoma160N.A. bone marrow cell multiple myeloma detection 161spermatozoa genome damage in reticulocyte detection 162N.A. keratinocytes classification of resting, proliferating, and differentiating cells 163carbonquantumdots488/500−560(DNA)543/570−650 (RNA)Caenorhabditis elegans (C. elegans) celldivisiontime-lapse imaging of chromatin and nucleoli during celldivision and C. elegans growth67DR1 560/600−650 (DNA)640/650−700 (RNA)HeLa cell high-content screening platform (cell cycle) 97TEG8-N14 640/650−720 (RNA)730/740−850 (DNA)NIH-3T3 cell necrosis detection (live)discrimination of apoptosis, necrosis,and necroptosis (fix)98ARPE-19 cell senescence at low and high passageaN.A.: not available.ACS Nano www.acsnano.org Reviewhttps://doi.org/10.1021/acsnano.5c22282ACS Nano 2026, 20, 6493−65146507www.acsnano.org?ref=pdfhttps://doi.org/10.1021/acsnano.5c22282?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ascan significantly influence the probe uptake, distribution, andbiological responses. Another critical requirement whenperforming cell culture for imaging is the imaging dura-tion.112,125 For long-term imaging, a 3D environment may benecessary to prevent alterations, such as nuclear flattening. Forshort-term tracking, factors such as nonspecific binding and theretention time of the probe to bind to specific subcellularorganelles must be carefully evaluated.Commercialization: Balancing Scalability, Quality Control,Safety, and CostTranslating imaging probes from the laboratory to commercialuse requires a balance between scalability, quality control,safety, and cost. Commercial fluorophores must meet stand-ards for consistency while remaining broadly accessible to bothacademic and industrial researchers. Ensuring this balance isessential for effective application in biological research.Achieving this balance is critical for the broad practicalapplication of whole RNA/DNA imaging (Table 2).■ PROSPECTIVE AND PROPOSALThis review focuses on RNA/DNA imaging, with the aim ofdeveloping practical molecular nanoarchitectonics. Althoughthis objective is fundamental, we examine whether usefultechnologies have been developed from the perspective of theuser and discuss the necessary future directions (Figure 12). Itis essential to develop molecular materials and techniques thatcan distinguish between DNA and RNA, image themaccurately in real time, and closely monitor their behavior.Therefore, there is an urgent need for a single-step, multiplexedRNA−DNA imaging technique that works under conditionscompatible with both RNA and DNA. Integrating both DNAand RNA information enables researchers to better understandhow structural genomic characteristics affect transcriptionalheterogeneity across different cell types and conditions whileminimizing artifacts. Among these methods, fluorescentimaging using chemical probes is the most convenient forlabeling RNA and DNA, as it can be performed withoutspecialized biological knowledge.Although perfect probes are rare, certain key designelements are essential for their optimal performance. It iscrucial to carefully select fluorescence excitation and emissionwavelengths that are tailored to the application. The brightnessof the probe after intracellular uptake is also important. Probeswith appropriate sensitivity are far more preferable to overlysensitive ones. For live-cell imaging, careful consideration mustbe given to the addition of cell-permeable substances.Multiplexed fluorescence imaging enables the simultaneousdetection of multiple intracellular targets and revealsintertarget relationships in a single step. In long-term imagingstudies, time-dependent specificity must be carefully consid-ered and evaluated. This is particularly important for achievinghigh target selectivity in the quantitative imaging of crowdedbiological environments.Recently, probes that meet many of these criteria have beendeveloped. For instance, TEG8-N14, which is discussed in thisreview, demonstrated the importance of N-rich structuralcompounds in enabling the simultaneous visualization of RNAand DNA using dual near-infrared excitation. This break-through not only minimizes phototoxicity and spectralcrosstalk due to NIR excitation but also demonstrates broadapplicability in measuring multiple types of cellular damage indifferent cell types. Dual-functional probes are essential foradvancing diagnostics and therapeutics. To improve theirfunctionality, strategies should focus on modifying theirchemical structures to enhance cell permeability, brightness,and performance.This review aims to provide an overview of the latestdevelopments in bioimaging, highlight currently overlookedintracellular imaging issues, and offer biological insights toencourage researchers in various fields to explore newstrategies related to their research. Our goal is to helpbiologists select the most appropriate approach for theirresearch and to support future researchers in developing next-Figure 12. Proposed development roadmap for commercial fluorophores based on the mechanism study and applied biological research.ACS Nano www.acsnano.org Reviewhttps://doi.org/10.1021/acsnano.5c22282ACS Nano 2026, 20, 6493−65146508https://pubs.acs.org/doi/10.1021/acsnano.5c22282?fig=fig12&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsnano.5c22282?fig=fig12&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsnano.5c22282?fig=fig12&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsnano.5c22282?fig=fig12&ref=pdfwww.acsnano.org?ref=pdfhttps://doi.org/10.1021/acsnano.5c22282?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-asgeneration RNA/DNA probes, from conceptual ideas tocommercialization, from a biologist’s perspective. We hopeto provide a useful guide. Below, we summarize therequirements for future RNA/DNA probe molecules.Ideal Criteria for Future RNA/DNA Probes(1) High sequence specificity� able to recognize one exactDNA or RNA sequence(2) NIR excitation and emission�avoid autofluorescenceand light scattering; low spectral overlap with commonlyused probes(3) Minimal light-induced chemical reactions�low photo-thermal effect and phototoxicity to avoid formingreactive byproducts under illumination. Such propertiesallow repeated imaging of both slow and fast biologicalprocesses(4) Single-parameter sensitivity�for example, responds onlyto a change in nucleic acid, not pH, aggregation, stableperformance, despite salt fluctuations or other environ-mental factors(5) High accumulation at the specific intracellular target�achieve a high signal-to-noise ratio to ensure compati-bility with any microscope, performs well even incrowded intracellular spaces(6) A stable signal over time�maintains sufficient signal atthe target during long experiments(7) High cell permeability�improves brightness and overallimaging performance(8) Low cross-reactivity with other probes�allows reliablecostaining(9) Small size�facilitates easy diffusion to tightly connectedcells.(10) High photostability�compatible with super-resolutionmicroscopy, including STED, which uses intense laserpower(11) Minimal perturbation� does not interfere with normalcellular processes or gene expression, minimal inter-action with membranes to avoid sticking to lipid bilayers(12) Good performance across cell types� broad applic-ability to various types of mammalian cells, and ifpossible, for plants, bacteria, and model organisms(13) Minimal precipitation�maintains solubility duringpreparation(14) Minimal-wash ability�minimizes any perturbance(15) High chemical stability�maintains its performance overtime in long-term storage conditions(16) Compatibility with flow cytometry�adaptable tovarious imaging platforms(17) Choose the correct fixation method� imaging out-comes must not be affected by the fixation process(18) Easy functionalization� can be linked to targetingligands, peptides, or antibodies(19) Long shelf life�stable during long-term storage(20) Scalable and cost-efficient synthesis�feasible for large-scale studies(21) High reproducibility across batches�consistent per-formanceOf course, ensuring reliable quantitative sensing capabilitiesduring dynamic therapeutic processes requires solid biologicalknowledge and accurate results. Other important factorsinclude the careful selection of each experimental componentfor characterization and the provision of a 3D environment forlong-term imaging in cell culture platforms. It is also essentialto balance scalability, quality control, safety, and cost forcommercializing these technologies. A comprehensive evalua-tion using in situ characterization techniques is also essential.Novel approaches, such as the introduction of AI technologyand direct molecular-level observation using probe microscopy,are expected to make a significant contribution. Nano-architectonics for molecular design and refinement ofevaluation methods will lead to the development of multi-plexed RNA-DNA imaging that functions in a single step underconditions compatible with both RNA and DNA. This willmake it more useful for users.■ AUTHOR INFORMATIONCorresponding AuthorsLinawati Sutrisno − International Center for Young Scientists(ICYS), National Institute for Materials Science, Tsukuba,Ibaraki 305-0044, Japan; orcid.org/0000-0003-3085-9660; Email: SUTRISNO.Linawati@nims.go.jpKatsuhiko Ariga − Research Center for MaterialsNanoarchitectonics (MANA), National Institute forMaterials Science, Tsukuba, Ibaraki 305-0044, Japan;Graduate School of Frontier Sciences, The University ofTokyo, Kashiwa 277-8561, Japan; orcid.org/0000-0002-2445-2955; Email: ARIGA.Katsuhiko@nims.go.jpAuthorKewei Sun − International Center for Young Scientists (ICYS),National Institute for Materials Science, Tsukuba, Ibaraki305-0044, Japan; Vacuum Interconnected NanotechWorkstation, Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, Suzhou 215123,China; orcid.org/0000-0002-1835-243XComplete contact information is available at:https://pubs.acs.org/10.1021/acsnano.5c22282Author ContributionsL.S. planned the manuscript, researched the literature, andwrote the original draft. S.K. co-wrote the original draft. K.A.discussed, reviewed, edited, co-wrote, and approved themanuscript.NotesThe authors declare no competing financial interest.■ ACKNOWLEDGMENTSThis work was supported in part by the Japan Society for thePromotion of Science (JSPS) KAKENHI Grant NumbersJP25H00898, JP25K23571, and JP24KF0089. L.S. acknowl-edges the support of the ICYS project.■ VOCABULARYAutofluorescence, is defined as the natural emission of light byendogenous fluorophores in biological systems when theyabsorb light; Absorbance coefficient, is a quantitative measure-ment that indicates how strongly a molecule absorbs light;Phototoxicity, refers to cellular damage caused by lightexposure that may alter the normal cellular behavior;Apoptosis, a programmed form of cell death that causeschanges in the cell morphology while maintaining membraneintegrity; Necrosis, an uncontrolled form of cell death thatshows loss of membrane integrity and release of intracellularcontentsACS Nano www.acsnano.org Reviewhttps://doi.org/10.1021/acsnano.5c22282ACS Nano 2026, 20, 6493−65146509https://pubs.acs.org/action/doSearch?field1=Contrib&text1="Linawati+Sutrisno"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdfhttps://orcid.org/0000-0003-3085-9660https://orcid.org/0000-0003-3085-9660mailto:SUTRISNO.Linawati@nims.go.jphttps://pubs.acs.org/action/doSearch?field1=Contrib&text1="Katsuhiko+Ariga"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdfhttps://orcid.org/0000-0002-2445-2955https://orcid.org/0000-0002-2445-2955mailto:ARIGA.Katsuhiko@nims.go.jphttps://pubs.acs.org/action/doSearch?field1=Contrib&text1="Kewei+Sun"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdfhttps://orcid.org/0000-0002-1835-243Xhttps://pubs.acs.org/doi/10.1021/acsnano.5c22282?ref=pdfwww.acsnano.org?ref=pdfhttps://doi.org/10.1021/acsnano.5c22282?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-as■ REFERENCES(1) Lee, S.; Huang, M.; Lee, J.; Choi, H.; Jo, I.-Y.; Na, H.; Lee, Y.;Youk, J. 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