Exploring chemistry with single-molecule and -particle fluorescence microscopy

Imaging of single-step chemical reactions and of single-catalytic turnovers at extended solid and molecular transition metal catalysts have been achieved.The techniques provide critical, otherwise inaccessible, information on reaction mechanisms, such as differentiation between catalytic phase (heterogenous and homogenous), observation of previously unidentified reaction intermediates, and differentiation between several plausible mechanistic pathways.Chemists can image in organic solvent and under air-free conditions to study and improve bench-scale preparative reactions. The evolution of single-molecule and -particle fluorescence microscopy imaging techniques for the investigation of chemical reactions has yielded achievements at all fronts, from synthetic organic chemistry to materials science. The removal of averaging effects uncovers unique, otherwise hidden reaction intermediates, reactivity, and kinetics. Thus, new insights can be obtained even for well-explored chemical reactions and processes. The aim of this article is to serve as a guide and a source of inspiration for scientists who wish to develop single-molecule and -particle fluorescence microscopy to answer questions in chemistry. We describe the current status of technical accomplishments in the field and point out technical and conceptual obstacles to overcome in the near future, with focus on improvement of bench-scale synthetic reactions. The evolution of single-molecule and -particle fluorescence microscopy imaging techniques for the investigation of chemical reactions has yielded achievements at all fronts, from synthetic organic chemistry to materials science. The removal of averaging effects uncovers unique, otherwise hidden reaction intermediates, reactivity, and kinetics. Thus, new insights can be obtained even for well-explored chemical reactions and processes. The aim of this article is to serve as a guide and a source of inspiration for scientists who wish to develop single-molecule and -particle fluorescence microscopy to answer questions in chemistry. We describe the current status of technical accomplishments in the field and point out technical and conceptual obstacles to overcome in the near future, with focus on improvement of bench-scale synthetic reactions. Synthetic chemists spend hours in front of the fume hood as they strive to achieve better reaction yields, control the chemo- and stereoselectivity of reactions, and develop sustainable processes. As a mechanistic organic chemistry and molecular catalysis research laboratory, we approach these fundamental challenges by studying reaction mechanisms: the elementary steps that transform simple starting materials to desirable, often more complex products. Yet, much of the mechanistic information we are interested in as synthetic chemists is hidden by ensemble averaging. Single-molecule fluorescence microscopy (see Glossary) and single-particle fluorescence microscopy remove that ensemble averaging, enabling the direct observation of reaction intermediates and spatial heterogeneities that would otherwise be obscured. Although subensemble fluorescence microscopy techniques have been employed to explore mechanisms in aqueous biological systems since the 1980s [1.Möckl L. Moerner W.E. Super-resolution microscopy with single molecules in biology and beyond−essentials, current trends, and future challenges.J. Am. Chem. Soc. 2020; 142: 17828-17844Crossref PubMed Scopus (49) Google Scholar], the adaptation to study chemical reactions was not straightforward and many challenges had to be overcome [2.Cordes T. Blum S.A. Opportunities and challenges in single-molecule and single-particle fluorescence microscopy for mechanistic studies of chemical reactions.Nat. Chem. 2013; 5: 993-999Crossref PubMed Scopus (115) Google Scholar]. For example, chemists had to translate microscopy techniques to achieve compatibility with the organic solvents [3.Esfandiari N.M. Blum S.A. Homogeneous vs heterogeneous polymerization catalysis revealed by single-particle fluorescence microscopy.J. Am. Chem. Soc. 2011; 133: 18145-18147Crossref PubMed Scopus (43) Google Scholar] and air-free techniques that are often critical for studying organic and organometallic reactions [4.Feng C. et al.Role of LiCl in generating soluble organozinc reagents.J. Am. Chem. Soc. 2016; 138: 11156-11159Crossref PubMed Scopus (54) Google Scholar,5.Menges J.A. et al.Kinetics of palladium(0)-allyl interactions in the Tsuji-Trost reaction, derived from single-molecule fluorescence microscopy.ChemCatChem. 2020; 12: 2630-2637Crossref Scopus (2) Google Scholar]. Equally important to these technical barriers, an initial lack of realization of shared scientific interests between single-molecule/-particle microscopy experts and synthetic/mechanistic chemists contributed to the relatively late blooming of the field. Now, many of these basic challenges have been solved. We posit that increased collaboration between researchers who understand and develop these microscopy methods and researchers with outstanding mechanistic questions in preparative synthetic chemistry and catalysis will lead to particularly exciting reaction insight, reaction development, and technical advances in microscopy imaging over the next decade (Figure 1). The purpose of this article is thus to serve as a guide for students and other new researchers who enter the field. It marks in condensed format what has already been accomplished and identifies key challenges that remain open, for inspiration. We provide a referenced list of operational methods that is easy for the new experimentalist to look up in Table 1 (Key table), for example, if they want to image in organic solvents, under air-free conditions, at elevated temperatures, or in a flow cell, without reinventing the wheel.Table 1Key table. A summary of technology/engineering demonstrations, types of reactions imaged, and mechanistic questions answeredReaction conditionsIn organic solvents [3.Esfandiari N.M. Blum S.A. Homogeneous vs heterogeneous polymerization catalysis revealed by single-particle fluorescence microscopy.J. Am. Chem. Soc. 2011; 133: 18145-18147Crossref PubMed Scopus (43) Google Scholar,44.Ng J.D. et al.Single-molecule investigation of initiation dynamics of an organometallic catalyst.J. Am. Chem. Soc. 2016; 138: 3876-3883Crossref PubMed Scopus (51) Google Scholar]Under air-free conditions (glovebox setup and adding solid reagents during imaging) [4.Feng C. et al.Role of LiCl in generating soluble organozinc reagents.J. Am. Chem. Soc. 2016; 138: 11156-11159Crossref PubMed Scopus (54) Google Scholar,5.Menges J.A. et al.Kinetics of palladium(0)-allyl interactions in the Tsuji-Trost reaction, derived from single-molecule fluorescence microscopy.ChemCatChem. 2020; 12: 2630-2637Crossref Scopus (2) Google Scholar]Imaging in flow cells [9.Xu W. et al.Single-molecule nanocatalysis reveals heterogeneous reaction pathways and catalytic dynamics.Nat. Mater. 2008; 7: 992-996Crossref PubMed Scopus (340) Google Scholar,32.Zhou X. et al.Size-dependent catalytic activity and dynamics of gold nanoparticles at the single-molecule level.J. Am. Chem. Soc. 2010; 132: 138-146Crossref PubMed Scopus (422) Google Scholar,52.Chen T. et al.Single-molecule nanocatalysis reveals catalytic activation energy of single nanocatalysts.J. Am. Chem. Soc. 2016; 138: 12414-12421Crossref PubMed Scopus (39) Google Scholar]Higher temperature imaging (diffusion at 135°C, chemical reaction at 45°C) [24.Flier B.M.I. et al.Heterogeneous diffusion in thin polymer films as observed by high-temperature single-molecule fluorescence microscopy.J. Am. Chem. Soc. 2012; 134: 480-488Crossref PubMed Scopus (76) Google Scholar,52.Chen T. et al.Single-molecule nanocatalysis reveals catalytic activation energy of single nanocatalysts.J. Am. Chem. Soc. 2016; 138: 12414-12421Crossref PubMed Scopus (39) Google Scholar]Types of reactions and processes imagedHydrolysis [6.Roeffaers M.B.J. et al.Spatially resolved observation of crystal-face-dependent catalysis by single turnover counting.Nature. 2006; 439: 572-575Crossref PubMed Scopus (372) Google Scholar]Proton-transfer [53.Ristanovic Z. et al.Single molecule nanospectroscopy visualizes proton-transfer processes within a zeolite crystal.J. Am. Chem. Soc. 2016; 138: 13586-13596Crossref PubMed Scopus (58) Google Scholar]Epoxidation [26.de Cremer G. et al.Heterogeneous catalysis high-resolution single-turnover mapping reveals intraparticle diffusion limitation in Ti-MCM-41-catalyzed epoxidation.Angew. Chem. Int. Ed. 2010; 49: 908-911Crossref PubMed Scopus (107) Google Scholar,33.Rybina A. et al.Single-molecule chemistry distinguishing alternative reaction pathways by single-molecule fluorescence spectroscopy.Angew. Chem. Int. Ed. 2013; 52: 6322-6325Crossref PubMed Scopus (51) Google Scholar]Solubilization [4.Feng C. et al.Role of LiCl in generating soluble organozinc reagents.J. Am. Chem. Soc. 2016; 138: 11156-11159Crossref PubMed Scopus (54) Google Scholar,36.Hanada E.M. et al.Mechanism of an elusive solvent effect in organozinc reagent synthesis.Chem. Eur. J. 2020; 26: 15094-15098Crossref PubMed Scopus (6) Google Scholar]Deallylation [5.Menges J.A. et al.Kinetics of palladium(0)-allyl interactions in the Tsuji-Trost reaction, derived from single-molecule fluorescence microscopy.ChemCatChem. 2020; 12: 2630-2637Crossref Scopus (2) Google Scholar]Redox (nanoparticle and photocatalysis) [10.Naito K. et al.Single-molecule observation of photocatalytic reaction in TiO 2 nanotube: importance of molecular transport through porous structures.J. Am. Chem. Soc. 2009; 131: 934-936Crossref PubMed Scopus (74) Google Scholar,11.Naito K. et al.Real-time single-molecule imaging of the spatial and temporal distribution of reactive oxygen species with fluorescent probes: applications to TiO 2 photocatalysts.J. Phys. Chem. C. 2008; 112: 1048-1059Crossref Scopus (77) Google Scholar,17.Wang W.-K. et al. Single-molecule and-particle probing crystal edge/corner as highly efficient photocatalytic sites on a single TiO 2 particle.Proc. Natl. Acad. Sci. U. S. A. 2019; 116: 18827-18833Crossref PubMed Scopus (30) Google Scholar,19.Ha J.W. et al.Super-resolution mapping of photogenerated electron and hole separation in single metal−semiconductor nanocatalysts.J. Am. Chem. Soc. 2014; 136: 1398-1408Crossref PubMed Scopus (123) Google Scholar, 20.Shen M. et al.Single-molecule colocalization of redox reactions on semiconductor photocatalysts connects surface heterogeneity and charge-carrier separation in bismuth oxybromide.J. Am. Chem. Soc. 2021; 143: 11393-11403Crossref PubMed Scopus (8) Google Scholar, 21.Shen M. et al.Nanoscale colocalization of fluorogenic probes reveals the role of oxygen vacancies in the photocatalytic activity of tungsten oxide nanowires.ACS Catal. 2020; 10: 2088-2099Crossref Scopus (30) Google Scholar,54.Christ T. et al.Watching the photo-oxidation of a single aromatic hydrocarbon molecule.Angew. Chem. Int. Ed. 2001; 40: 4192-4195Crossref PubMed Scopus (97) Google Scholar]Cross-coupling [46.Costa P. et al.Real-time fluorescence imaging of a heterogeneously catalysed Suzuki–Miyaura reaction.Nat. Catal. 2020; 3: 427-437Crossref Scopus (23) Google Scholar]Oligomerization [14.Ristanovic´ Z.R. et al.Heterogeneous catalysis hot paper high-resolution single-molecule fluorescence imaging of zeolite aggregates within real-life fluid catalytic cracking particles.Angew. Chem. Int. Ed. 2015; 54: 1836-1840Crossref PubMed Scopus (75) Google Scholar]Polymerization [40.Hensle E.M. Blum S.A. Phase separation polymerization of dicyclopentadiene characterized by in operando fluorescence microscopy.J. Am. Chem. Soc. 2013; 135: 12324-12328Crossref PubMed Scopus (31) Google Scholar, 41.Blum S.A. Easter Q.T. Single turnover at molecular polymerization catalysts reveals spatiotemporally resolved reactions.Angew. Chem. Int. Ed. 2017; 56: 13772-13775Crossref PubMed Scopus (23) Google Scholar, 42.Easter Q.T. et al.Single-polymer−particle growth kinetics with molecular catalyst speciation and single-turnover imaging.ACS Catal. 2019; 9: 3375-3383Crossref Scopus (10) Google Scholar,55.Easter Q.T. Blum S.A. Kinetics of the same reaction over nine orders of magnitude in concentration: when are unique subensemble and single-turnover reactivity displayed?.Angew. Chem. Int. Ed. 2018; 57: 12027-12032Crossref PubMed Scopus (14) Google Scholar]Hydrogenation [56.Wang B. et al.Mechanistic insights on the semihydrogenation of alkynes over different nanostructured photocatalysts.ACS Catal. 2021; 11: 4230-4238Crossref Scopus (1) Google Scholar]Cycloaddition [47.Wang B. et al.Click chemistry: mechanistic insights into the role of amines using single-molecule spectroscopy.ACS Catal. 2017; 7: 8487-8492Crossref Scopus (9) Google Scholar, 48.Decan M.R. et al.Copper nanoparticle heterogeneous catalytic "click" cycloaddition confirmed by single-molecule spectroscopy.Nat. Commun. 2014; 5: 4612-4619Crossref PubMed Scopus (103) Google Scholar, 49.Decan M.R. Scaiano J.C. Study of single catalytic events at copper-in-charcoal: localization of click activity through subdiffraction observation of single catalytic events.J. Phys. Chem. Lett. 2015; 6: 4049-4053Crossref PubMed Scopus (13) Google Scholar, 50.Wang B. et al.From the molecule to the mole: improving heterogeneous copper catalyzed click chemistry using single molecule spectroscopy.Chem. Commun. 2017; 53: 328-331Crossref Scopus (7) Google Scholar]Condensation [34.Gehlen M.H. et al.Single-molecule observations provide mechanistic insights into bimolecular Knoevenagel amino catalysis.J. Phys. Chem. Lett. 2020; 11: 9714-9724Crossref PubMed Scopus (2) Google Scholar,57.Hodgson G.K. et al.Dye synthesis in the Pechmann reaction: catalytic behaviour of samarium oxide nanoparticles studied using single molecule fluorescence microscopy.Chem. Sci. 2016; 7: 1314-1321Crossref PubMed Google Scholar]Corrosion [15.Saini A. et al.Fluorophores “turned-on” by corrosion reactions can be detected at the single-molecule level.ACS Appl. Mater. Interfaces. 2021; 13: 2000-2006Crossref PubMed Scopus (8) Google Scholar,16.Saini A. et al.Investigation of fluorophores for single-molecule detection of anodic corrosion redox reactions.MRS Commun. 2021; (Published online September 23, 2021)https://doi.org/10.1557/S43579-021-00096-YCrossref Scopus (1) Google Scholar]Molecular metal–ligand exchange/dissociation [37.Lim S.-G. Blum S.A. A general fluorescence resonance energy transfer (FRET) method for observation and quantification of organometallic complexes under reaction conditions.Organometallics. 2009; 28: 4643-4645Crossref Scopus (19) Google Scholar, 38.Esfandiari N.M. et al.Single-molecule imaging of platinum ligand exchange reaction reveals reactivity distribution.J. Am. Chem. Soc. 2010; 132: 15167-15169Crossref PubMed Scopus (37) Google Scholar, 39.Esfandiari N.M. et al.Real-time imaging of platinum-sulfur ligand exchange reactions at the single-molecule level via a general chemical technique.Organometallics. 2011; 30: 2901-2907Crossref Scopus (23) Google Scholar,44.Ng J.D. et al.Single-molecule investigation of initiation dynamics of an organometallic catalyst.J. Am. Chem. Soc. 2016; 138: 3876-3883Crossref PubMed Scopus (51) Google Scholar]Oxidative addition [4.Feng C. et al.Role of LiCl in generating soluble organozinc reagents.J. Am. Chem. Soc. 2016; 138: 11156-11159Crossref PubMed Scopus (54) Google Scholar,35.Jess K. et al.Microscopy reveals: impact of lithium salts on elementary steps predicts organozinc reagent synthesis and structure.J. Am. Chem. Soc. 2019; 141: 9879-9884Crossref PubMed Scopus (13) Google Scholar,46.Costa P. et al.Real-time fluorescence imaging of a heterogeneously catalysed Suzuki–Miyaura reaction.Nat. Catal. 2020; 3: 427-437Crossref Scopus (23) Google Scholar]Mechanistic questions answeredIdentification of previously unknown organic and organometallic intermediates [33.Rybina A. et al.Single-molecule chemistry distinguishing alternative reaction pathways by single-molecule fluorescence spectroscopy.Angew. Chem. Int. Ed. 2013; 52: 6322-6325Crossref PubMed Scopus (51) Google Scholar,34.Gehlen M.H. et al.Single-molecule observations provide mechanistic insights into bimolecular Knoevenagel amino catalysis.J. Phys. Chem. Lett. 2020; 11: 9714-9724Crossref PubMed Scopus (2) Google Scholar]Origins of solvent effects [36.Hanada E.M. et al.Mechanism of an elusive solvent effect in organozinc reagent synthesis.Chem. Eur. J. 2020; 26: 15094-15098Crossref PubMed Scopus (6) Google Scholar]Roles of reagents [4.Feng C. et al.Role of LiCl in generating soluble organozinc reagents.J. Am. Chem. Soc. 2016; 138: 11156-11159Crossref PubMed Scopus (54) Google Scholar,34.Gehlen M.H. et al.Single-molecule observations provide mechanistic insights into bimolecular Knoevenagel amino catalysis.J. Phys. Chem. Lett. 2020; 11: 9714-9724Crossref PubMed Scopus (2) Google Scholar,35.Jess K. et al.Microscopy reveals: impact of lithium salts on elementary steps predicts organozinc reagent synthesis and structure.J. Am. Chem. Soc. 2019; 141: 9879-9884Crossref PubMed Scopus (13) Google Scholar]Origin of reaction suppression and subsequent macroscale improvement [50.Wang B. et al.From the molecule to the mole: improving heterogeneous copper catalyzed click chemistry using single molecule spectroscopy.Chem. Commun. 2017; 53: 328-331Crossref Scopus (7) Google Scholar,58.Jess K. et al.Origins of batch-to-batch variation: organoindium reagents from indium metal.Organometallics. 2020; 39: 2575-2579Crossref PubMed Scopus (2) Google Scholar]Location of catalysis in heterogeneous systems [3.Esfandiari N.M. Blum S.A. Homogeneous vs heterogeneous polymerization catalysis revealed by single-particle fluorescence microscopy.J. Am. Chem. Soc. 2011; 133: 18145-18147Crossref PubMed Scopus (43) Google Scholar,46.Costa P. et al.Real-time fluorescence imaging of a heterogeneously catalysed Suzuki–Miyaura reaction.Nat. Catal. 2020; 3: 427-437Crossref Scopus (23) Google Scholar,48.Decan M.R. et al.Copper nanoparticle heterogeneous catalytic "click" cycloaddition confirmed by single-molecule spectroscopy.Nat. Commun. 2014; 5: 4612-4619Crossref PubMed Scopus (103) Google Scholar]Phases of active catalysts [6.Roeffaers M.B.J. et al.Spatially resolved observation of crystal-face-dependent catalysis by single turnover counting.Nature. 2006; 439: 572-575Crossref PubMed Scopus (372) Google Scholar,9.Xu W. et al.Single-molecule nanocatalysis reveals heterogeneous reaction pathways and catalytic dynamics.Nat. Mater. 2008; 7: 992-996Crossref PubMed Scopus (340) Google Scholar]Active catalytic domains in zeolite/solid particles [13.Buurmans I.L.C. et al.Catalytic activity in individual cracking catalyst particles imaged throughout different life stages by selective staining.Nat. Chem. 2011; 3: 862-867Crossref PubMed Scopus (110) Google Scholar,14.Ristanovic´ Z.R. et al.Heterogeneous catalysis hot paper high-resolution single-molecule fluorescence imaging of zeolite aggregates within real-life fluid catalytic cracking particles.Angew. Chem. Int. Ed. 2015; 54: 1836-1840Crossref PubMed Scopus (75) Google Scholar,59.Filez M. et al.Chemical imaging of hierarchical porosity formation within a zeolite crystal visualized by small-angle X-ray scattering and in-situ fluorescence microscopy.Angew. Chem. Int. Ed. 2021; 60: 13803-13806Crossref PubMed Scopus (2) Google Scholar]Nano-confinement effects [18.Dong B. et al.Deciphering nanoconfinement effects on molecular orientation and reaction intermediate by single molecule imaging.Nat. Commun. 2019; 10: 4815-4820Crossref PubMed Scopus (20) Google Scholar]Selectivity of individual molecular catalysts [43.Garcia A. et al.Does selectivity of molecular catalysts change with time? Polymerization imaged by single-molecule spectroscopy.Angew. Chem. Int. Ed. 2021; 60: 1550-1555Crossref PubMed Scopus (6) Google Scholar] and solid catalysts [21.Shen M. et al.Nanoscale colocalization of fluorogenic probes reveals the role of oxygen vacancies in the photocatalytic activity of tungsten oxide nanowires.ACS Catal. 2020; 10: 2088-2099Crossref Scopus (30) Google Scholar,60.Shen M. et al.Competing activation and deactivation mechanisms in photodoped bismuth oxybromide nanoplates probed by single-molecule fluorescence imaging.J. Phys. Chem. Lett. 2020; 11: 5219-5227Crossref PubMed Scopus (8) Google Scholar]Surface dissociation/association kinetics [9.Xu W. et al.Single-molecule nanocatalysis reveals heterogeneous reaction pathways and catalytic dynamics.Nat. Mater. 2008; 7: 992-996Crossref PubMed Scopus (340) Google Scholar,32.Zhou X. et al.Size-dependent catalytic activity and dynamics of gold nanoparticles at the single-molecule level.J. Am. Chem. Soc. 2010; 132: 138-146Crossref PubMed Scopus (422) Google Scholar,52.Chen T. et al.Single-molecule nanocatalysis reveals catalytic activation energy of single nanocatalysts.J. Am. Chem. Soc. 2016; 138: 12414-12421Crossref PubMed Scopus (39) Google Scholar]Surface and pore diffusion kinetics distributions [10.Naito K. et al.Single-molecule observation of photocatalytic reaction in TiO 2 nanotube: importance of molecular transport through porous structures.J. Am. Chem. Soc. 2009; 131: 934-936Crossref PubMed Scopus (74) Google Scholar,24.Flier B.M.I. et al.Heterogeneous diffusion in thin polymer films as observed by high-temperature single-molecule fluorescence microscopy.J. Am. Chem. Soc. 2012; 134: 480-488Crossref PubMed Scopus (76) Google Scholar, 25.Hendriks F.C. et al.Single-molecule fluorescence microscopy reveals local diffusion coefficients in the pore network of an individual catalyst particle.J. Am. Chem. Soc. 2017; 139: 13632-13635Crossref PubMed Scopus (45) Google Scholar, 26.de Cremer G. et al.Heterogeneous catalysis high-resolution single-turnover mapping reveals intraparticle diffusion limitation in Ti-MCM-41-catalyzed epoxidation.Angew. Chem. Int. Ed. 2010; 49: 908-911Crossref PubMed Scopus (107) Google Scholar, 27.Yuan T. et al.Vertical diffusion of ions within single particles during electrochemical charging.ACS Nano. 2021; 15: 3522-3528Crossref PubMed Scopus (7) Google Scholar]Catalyst speciation [42.Easter Q.T. et al.Single-polymer−particle growth kinetics with molecular catalyst speciation and single-turnover imaging.ACS Catal. 2019; 9: 3375-3383Crossref Scopus (10) Google Scholar] Open table in a new tab In order to study chemical reactions at this high level of sensitivity and spatial resolution, it is required to observe elementary, single-step, reactions and to know with certainty which step in the chemical process is being observed (or at least to be able to narrow down the options to a couple of plausible choices). As part of this certainty, knowledge of the molecular structures of the observed starting materials, intermediates, or products is critical. In our assessment, the central breakthrough in this ability occurred in 2006, when Roeffaers and Hofkens demonstrated hydrolysis of organic molecules involving single catalytic turnovers on inorganic crystal faces [6.Roeffaers M.B.J. et al.Spatially resolved observation of crystal-face-dependent catalysis by single turnover counting.Nature. 2006; 439: 572-575Crossref PubMed Scopus (372) Google Scholar]. This was followed by contributions from Blum, who demonstrated the imaging of the first organometallic complex and transition metal ligand exchange at the single-molecule levels [7.Canham S.M. et al.Toward the single-molecule investigation of organometallic reaction mechanisms: single-molecule imaging of fluorophore-tagged palladium(II) complexes.Organometallics. 2008; 27: 2172-2175Crossref Scopus (30) Google Scholar,8.Easter Q.T. Blum S.A. Organic and organometallic chemistry at the single-molecule, -particle, and -molecular-catalyst-turnover level by fluorescence microscopy.Acc. Chem. Res. 2019; 8: 2244-2255Crossref Scopus (16) Google Scholar], from Chen, who demonstrated turnover kinetics at catalytic gold nanoparticles [9.Xu W. et al.Single-molecule nanocatalysis reveals heterogeneous reaction pathways and catalytic dynamics.Nat. Mater. 2008; 7: 992-996Crossref PubMed Scopus (340) Google Scholar], and from Majima, who demonstrated single turnovers in photocatalytic systems [10.Naito K. et al.Single-molecule observation of photocatalytic reaction in TiO 2 nanotube: importance of molecular transport through porous structures.J. Am. Chem. Soc. 2009; 131: 934-936Crossref PubMed Scopus (74) Google Scholar,11.Naito K. et al.Real-time single-molecule imaging of the spatial and temporal distribution of reactive oxygen species with fluorescent probes: applications to TiO 2 photocatalysts.J. Phys. Chem. C. 2008; 112: 1048-1059Crossref Scopus (77) Google Scholar]. From an operations standpoint, these experiments demonstrated reactions in flow cells, manipulations under air-free conditions, and spatial determinations of locations of reactions and molecules (Table 1). These studies became the foundations for more advanced single-molecule experiments in chemistry. Many industrially relevant processes occur on solid/liquid interfaces of heterogeneous systems [12.End N. Schöning K.-U. Immobilized catalysts in industrial research and application.in: Kirschning A. Immobilized Catalysts: Solid Phases, Immobilization and Applications. Springer, 2004: 241-271Crossref Google Scholar]. Such systems are well-suited for investigation by single-molecule (reactant or product) imaging and single-particle (solid) imaging because the reactants become immobilized or otherwise diffuse slowly when confined by the solids. This slow diffusion enables their imaging by fluorescence microscopy. Insights into fluid cracking catalysis using catalytic zeolite particle systems [13.Buurmans I.L.C. et al.Catalytic activity in individual cracking catalyst particles imaged throughout different life stages by selective staining.Nat. Chem. 2011; 3: 862-867Crossref PubMed Scopus (110) Google Scholar,14.Ristanovic´ Z.R. et al.Heterogeneous catalysis hot paper high-resolution single-molecule fluorescence imaging of zeolite aggregates within real-life fluid catalytic cracking particles.Angew. Chem. Int. Ed. 2015; 54: 1836-1840Crossref PubMed Scopus (75) Google Scholar] and corrosion detection on iron cathodes [15.Saini A. et al.Fluorophores “turned-on” by corrosion reactions can be detected at the single-molecule level.ACS Appl. Mater. Interfaces. 2021; 13: 2000-2006Crossref PubMed Scopus (8) Google Scholar,16.Saini A. et al.Investigation of fluorophores for single-molecule detection of anodic corrosion redox reactions.MRS Commun. 2021; (Published online September 23, 2021)https://doi.org/10.1557/S43579-021-00096-YCrossref Scopus (1) Google Scholar] are example systems where unique insight has been obtained. The spatiotemporal resolution of fluorescence microscopy provides exquisite 2D and 3D reactivity maps of the location of catalytic reactions on and within these catalytic inorganic materials [11.Naito K. et al.Real-time single-molecule imaging of the spatial and temporal distribution of reactive oxygen species with fluorescent probes: applications to TiO 2 photocatalysts.J. Phys. Chem. C. 2008; 112: 1048-1059Crossref Scopus (77) Google Scholar,17.Wang W.-K. et al. Single-molecule and-particle probing crystal edge/corner as highly efficient photocatalytic sites on a single TiO 2 particle.Proc. Natl. Acad. Sci. U. S. A. 2019; 116: 18827-18833Crossref PubMed Scopus (30) Google Scholar, 18.Dong B. et al.Deciphering nanoconfinement effects on molecular orientation and reaction intermediate by single molecule imaging.Nat. Commun. 2019; 10: 4815-4820Crossref PubMed Scopus (20) Google Scholar, 19.Ha J.W. et al.Super-resolution mapping of photogenerated electron and hole separation in single metal−semiconductor nanocatalysts.J. Am. Chem. Soc. 2014; 136: 1398-1408Crossref PubMed Scopus (123) Google Scholar, 20.Shen M. et al.Single-molecule colocalization of redox reactions on semiconductor photocatalysts connects surface heterogeneity and charge-carrier separation in bismuth oxybromide.J. Am. Chem. Soc. 2021; 143: 11393-11403Crossref PubMed Scopus (8) Google Scholar, 21.Shen M. et al.Nanoscale colocalization of fluorogenic probes reveals the role of oxygen vacancies in the photocatalytic activity of tungsten oxide nanowires.ACS Catal. 2020; 10: 2088-2099Crossref Scopus (30) Google Scholar, 22.Evans R.C. et al.Influence of single-nanoparticle electrochromic dynamics on the durability and speed of smart windows.Proc. Natl. Acad. Sci. U. S. A. 2019; 116: 12666-12671Crossref PubMed Scopus (28) Google Scholar, 23.Wang W. Imaging the chemical activity of single nanoparticles with optical microscopy.Chem. Soc. Rev. 2018; 47: 2485-2508Crossref PubMed Google Scholar]. Super-resolution and superlocalization techniques are the standard in these systems, and they enable tracking of small organic molecule and polymer diffusion on and inside of catalytic particles, porous materials, and polymer films [10.Naito K. et al.Single-molecule observation of photocatalytic reaction in TiO 2 nanotube: importance of molecular transport through porous structures.J. Am. Chem. Soc. 2009; 131: 934-936Crossref PubMed Scopus (74) Google Scholar,24.Flier B.M.I. et al.Heterogeneous diffusion in thin polymer films as observed by high-temperature single-molecule fluorescence microscopy.J. Am. Chem. Soc. 2012; 134: 480-488Crossref PubMed Scopus (76) Google Scholar, 25.Hendriks F.C. et al.Single-molecule fluorescence microscopy reveals local diffusion coefficients in the pore network of an individual catalyst particle.J. Am. Chem. Soc. 2017; 139: 13632-13635Crossref PubMed Scopus (45) Google Scholar, 26.de Cremer G. et al.Heterogeneous catalysis high-resolution single-turnover mapping reveals intraparticle diffusion limitation in Ti-MCM-41-catalyzed epoxidation.Angew. Chem. Int.