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Single-molecule localization microscopy (SMLM) has revolutionized our ability to visualize cellular structures, offering unprecedented detail. However, the intricate biophysical principles that underlie SMLM can be daunting for newcomers, particularly undergraduate and graduate students. To address this challenge, we introduce the fundamental concepts of SMLM, providing a solid theoretical foundation. In addition, we have developed an intuitive graphical interface APP that simplifies these core concepts, making them more accessible for students. This APP clarifies how super-resolved images are fitted and highlights the crucial factors determining image quality. Our approach deepens students' understanding of SMLM by combining theoretical instruction with practical learning. This development equips them with the skills to carry out single-molecule super-resolved experiments and explore the microscopic world beyond the diffraction limit.
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The introduction of super-resolution microscopy (SRM) has significantly advanced our understanding of cellular and molecular dynamics, offering a detailed view previously beyond our reach. Implementing SRM in biophysical research, however, presents numerous challenges. This review addresses the crucial aspects of utilizing SRM effectively, from selecting appropriate fluorophores and preparing samples to analyzing complex data sets. We explore recent technological advancements and methodological improvements that enhance the capabilities of SRM. Emphasizing the integration of SRM with other analytical methods, we aim to overcome inherent limitations and expand the scope of biological insights achievable. By providing a comprehensive guide for choosing the most suitable SRM methods based on specific research objectives, we aim to empower researchers to explore complex biological processes with enhanced precision and clarity, thereby advancing the frontiers of biophysical research.
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Deciphering the oligomeric state of proteins within cells is pivotal to understanding their role in intricate cellular processes. With the recent advances in single-molecule localization microscopy, previous efforts have harnessed protein location density approaches, coupled with simulations, to extract membrane protein oligomeric states in cells, highlighting the value of such techniques. However, a comprehensive theoretical approach that can be universally applied across different proteins (e.g., membrane and cytosolic proteins) remains elusive. Here, we introduce the theoretical probability of neighbor density (PND) as a robust tool to discern protein oligomeric states in cellular environments. Utilizing our approach, the theoretical PND was validated against simulated data for both membrane and cytosolic proteins, consistently aligning with experimental baselines for membrane proteins. This congruence was maintained even when adjusting for protein concentrations or exploring proteins of various oligomeric states. The strength of our method lies not only in its precision but also in its adaptability, accommodating diverse cellular protein scenarios without compromising the accuracy. The development and validation of the theoretical PND facilitate accurate protein oligomeric state determination and bolster our understanding of protein-mediated cellular functions.
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Proteínas de Membrana , Imagem Individual de Molécula , Proteínas de Membrana/metabolismo , Imagem Individual de Molécula/métodos , Citosol/metabolismoRESUMO
Studies of organometallic reactions in living cells commonly rely on ensemble-averaged measurements, which can obscure the detection of reaction dynamics or location-specific behavior. This information is necessary to guide the design of bioorthogonal catalysts with improved biocompatibility, activity, and selectivity. By leveraging the high spatial and temporal resolution of single-molecule fluorescence microscopy, we have successfully captured single-molecule events promoted by Ru complexes inside live A549 human lung cells. By observing individual allylcarbamate cleavage reactions in real-time, our results revealed that they occur with greater frequency inside the mitochondria than in the non-mitochondria regions. The estimated turnover frequency of the Ru complexes was at least 3-fold higher in the former than the latter. These results suggest that organelle specificity is a critical factor to consider in intracellular catalyst design, such as in developing metallodrugs for therapeutic applications.
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Compostos Organometálicos , Rutênio , Humanos , Mitocôndrias , Microscopia de Fluorescência , Catálise , Compostos Organometálicos/farmacologiaRESUMO
The protein Cu/Zn superoxide dismutase (SOD1) is known to function as a dimer, but its concentration in cells (â¼50 µM) and the dimerization constant (K d of 500 µM) results suggest that it exists in a monomer-dimer equilibrium. It is unclear how the oligomeric state of SOD1 changes when cells are initially exposed to high levels of extracellular oxidative stress. To address this problem, we introduced the single-molecule fluorescence anisotropy (smFA) assay to explore SOD1 oligomeric states in live COS7 cells. smFA specifically probes the fluorescence polarization changes caused by molecular rotations where the fast-rotating molecules (either due to smaller hydrodynamic volume or less viscous environments) deteriorate the emission polarization and thus lower the anisotropy. After validating that smFA is effective in distinguishing monomeric and dimeric fluorescence proteins, we overexpressed SOD1 in live COS7 cells and investigated how its oligomeric state changes under basal, 2 h, and 24 h 100 µM H2O2 treatments. We found that treating cells with H2O2 promotes SOD1 dimerization and decreases cellular viscosity in 2 h. Interestingly, prolonged H2O2 treatments show similar results as the basal conditions, indicating that cells return to a steady state similar to the basal state after 24 h, despite the presence of H2O2. Our results demonstrate that SOD1 changes its oligomeric state equilibrium in response to extracellular oxidative stresses. smFA will open new opportunities to explore the relationship between the SOD1 oligomer state and its H2O2-based signaling and transcription regulation roles.
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Recent studies have uncovered the therapeutic potential of elesclomol (ES), a copper-ionophore, for copper deficiency disorders. However, we currently do not understand the mechanism by which copper brought into cells as ES-Cu(II) is released and delivered to cuproenzymes present in different subcellular compartments. Here, we have utilized a combination of genetic, biochemical, and cell-biological approaches to demonstrate that intracellular release of copper from ES occurs inside and outside of mitochondria. The mitochondrial matrix reductase, FDX1, catalyzes the reduction of ES-Cu(II) to Cu(I), releasing it into mitochondria where it is bioavailable for the metalation of mitochondrial cuproenzyme- cytochrome c oxidase. Consistently, ES fails to rescue cytochrome c oxidase abundance and activity in copper-deficient cells lacking FDX1. In the absence of FDX1, the ES-dependent increase in cellular copper is attenuated but not abolished. Thus, ES-mediated copper delivery to nonmitochondrial cuproproteins continues even in the absence of FDX1, suggesting alternate mechanism(s) of copper release. Importantly, we demonstrate that this mechanism of copper transport by ES is distinct from other clinically used copper-transporting drugs. Our study uncovers a unique mode of intracellular copper delivery by ES and may further aid in repurposing this anticancer drug for copper deficiency disorders.
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Cobre , Complexo IV da Cadeia de Transporte de Elétrons , Hidrazinas , Ionóforos , Ferredoxinas/metabolismoRESUMO
Excess intracellular Cu perturbs cellular redox balance and thus causes diseases. However, the relationship between cellular redox status and Cu homeostasis and how such an interplay is coordinated within cellular compartments has not yet been well established. Using combined approaches of organelle-specific redox sensor Grx1-roGFP2 and non-targeted proteomics, we investigate the real-time Cu-dependent antioxidant defenses of mitochondria and cytosol in live HEK293 cells. The Cu-dependent real-time imaging experiments show that CuCl2 treatment results in increased oxidative stress in both cytosol and mitochondria. In contrast, subsequent excess Cu removal by bathocuproine sulfonate, a Cu chelating reagent, lowers oxidative stress in mitochondria but causes even higher oxidative stress in the cytosol. The proteomic data reveal that several mitochondrial proteins, but not cytosolic ones, undergo significant abundance change under Cu treatments. The proteomic analysis also shows that proteins with significant changes are related to mitochondrial oxidative phosphorylation and glutathione synthesis. The differences in redox behaviors and protein profiles in different cellular compartments reveal distinct mitochondrial and cytosolic response mechanisms upon Cu-induced oxidative stress. These findings provide insights into how redox and Cu homeostasis interplay by modulating specific protein expressions at the subcellular levels, shedding light on understanding the effects of Cu-induced redox misregulation on the diseases.
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Antioxidantes , Proteômica , Humanos , Antioxidantes/farmacologia , Células HEK293 , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Fluorescência Verde/farmacologia , Oxirredução , Mitocôndrias/metabolismo , Estresse Oxidativo , Glutationa/metabolismoRESUMO
Copper transporter 1 (CTR1) is the major membrane protein responsible for cellular copper (Cu) uptake and mediates cellular copper homeostasis. To elucidate CTR1's behavior using imaging approaches, we generated a homozygous knock-in human embryonic stem cell (hESC) clone expressing photoconvertible fluorescence protein mEos4b-tagged endogenous CTR1 using CRISPR-Cas9 mediated homologous recombination. The engineered cells express functional CTR1-mEos4b fusion and have normal stem cell morphology. They remain pluripotent and can be differentiated into all three germ layers in vitro. This resource allows the study of CTR1 at an endogenous level in different cellular contexts using microscopy.
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Proteínas de Transporte de Cátions , Células-Tronco Embrionárias Humanas , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Linhagem Celular , Cobre/metabolismo , Transportador de Cobre 1 , Homozigoto , Células-Tronco Embrionárias Humanas/metabolismo , HumanosRESUMO
Imbalanced copper homeostasis and perturbation of membrane trafficking are two common symptoms that have been associated with the pathogenesis of neurodegenerative and neurodevelopmental diseases. Accumulating evidence from biophysical, cellular and in vivo studies suggest that membrane trafficking orchestrates both copper homeostasis and neural functions-however, a systematic review of how copper homeostasis and membrane trafficking interplays in neurons remains lacking. Here, we summarize current knowledge of the general trafficking itineraries for copper transporters and highlight several critical membrane trafficking regulators in maintaining copper homeostasis. We discuss how membrane trafficking regulators may alter copper transporter distribution in different membrane compartments to regulate intracellular copper homeostasis. Using Parkinson's disease and MEDNIK as examples, we further elaborate how misregulated trafficking regulators may interplay parallelly or synergistically with copper dyshomeostasis in devastating pathogenesis in neurodegenerative diseases. Finally, we explore multiple unsolved questions and highlight the existing challenges to understand how copper homeostasis is modulated through membrane trafficking.
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Proteínas de Transporte de Cobre/metabolismo , Cobre/metabolismo , Doenças do Sistema Nervoso/metabolismo , Sistema Nervoso/metabolismo , Animais , Regulação da Expressão Gênica , Homeostase , Humanos , Doença de Parkinson/metabolismo , Transdução de SinaisRESUMO
Superoxide Dismutase 1 (SOD1) is an antioxidant enzyme that protects the cells from radical oxygen species. To study the behavior of endogenous SOD1 under a microscope, we genetically modified H1 human embryonic stem cells (hESCs) to express SOD1 fused with a SNAP-tag, a protein tag that can be covalently labeled with a variety of synthetic probes. The engineered homozygous clone expressing SOD1-SNAP fusion proteins has normal stem cell morphology and karyotype, expresses pluripotency markers, and can be differentiated into all three germ layers in vitro, providing a versatile platform for imaging-based studies of SOD1.
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Células-Tronco Embrionárias Humanas , Linhagem Celular , Células Cultivadas , Humanos , Superóxido Dismutase/genética , Superóxido Dismutase-1/genéticaRESUMO
Protein organization modification plays a vital role in initiating signaling pathways, transcriptional regulation, and cell apoptosis regulation. Simultaneous quantification of oligomeric state and cellular parameters in the same cell, even though challenging, is required to understand their correlation at the molecular level. Recent advances of fluorescence protein and single-molecule localization microscopy enables the determination of localizations and oligomeric states of target proteins in cells. We reviewed the fluorescence intensity-based, localization-based, and photophysical property-based approaches for in-cell quantification of protein oligomeric stoichiometry. We discussed their working principles, applications, advantages, and limitations. These results also imply the combination of methodologies targeting different biological parameters at the single-cell level is essential to uncover the structure-function relationship at the molecular level.
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Proteínas , Imagem Individual de Molécula , Microscopia de FluorescênciaRESUMO
The ability to extract kinetic interaction parameters from single-molecule fluorescence resonance energy transfer trajectories without the need for solving complex single-molecule differential equations has the potential to address some of the critical biophysical questions. Here, we provide a noise-free single-molecule interaction simulation (SMIS) tool to give the expected dwell-time distributions and relative populations of each FRET level based on the assigned kinetic model and to dissect kinetic interaction parameters from single-molecule FRET trajectories. The method provides the expected dwell-time distributions, average transition rates, and relative populations of each FRET level based on the assigned kinetic model. By comparing with ground truth data and experimental data, we demonstrated that SMIS is useful to quantify the interaction kinetic rate constants without using the traditional single-molecule analytical solution approach.
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Imagem Individual de Molécula/métodos , Dissecação , Transferência Ressonante de Energia de Fluorescência , Cinética , Modelos Químicos , TermodinâmicaRESUMO
ATOX1 is a copper chaperone involved in intracellular copper homeostasis, cell proliferation, and tumor progression. To investigate the physiologically relevant molecular mechanism of ATOX1 by using imaging-based approaches, we genetically modified ATOX1 in H1 hESCs to express mCherry-ATOX1 fusion protein under endogenous regulatory machinery. The fluorescence engineered hESC clone maintains characteristic stem cell features and can differentiate to all three germ layers, serving as a unique tool to dissect the role of ATOX1 in various cellular processes.
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Técnicas de Cultura de Células/métodos , Proteínas de Transporte de Cobre/genética , Corantes Fluorescentes/metabolismo , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/metabolismo , Chaperonas Moleculares/genética , Sequência de Bases , Humanos , Masculino , Reprodutibilidade dos TestesRESUMO
Transitions between different oligomeric states of membrane proteins are essential for proper cellular functions. However, the quantification of their oligomeric states in cells is technically challenging. Here we developed a new method to quantify oligomeric state(s) of highly expressed membrane proteins using the probability density function of molecule density ( PDFMD) calculated from super-resolution localizations. We provided the theoretical model of PDFMD, discussed the effects of protein concentration, cell geometry, and photophysics of fluorescent proteins on PDFMD, and provided experimental criteria for proper quantification of oligomeric states. This method was further validated using simulated single-molecule fluorescent movies and applied to two membrane proteins, UhpT and SbmA in E. coli. The study shows that PDFMD is useful in quantifying oligomeric states of membrane proteins in cells that can help in understanding cellular tasks. Potential applications to proteins with higher oligomeric states under high concentration and limitations of our methodology were also discussed.
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Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Transporte de Monossacarídeos/metabolismo , Escherichia coli/química , Proteínas de Escherichia coli/química , Proteínas de Membrana Transportadoras/química , Microscopia , Modelos Químicos , Modelos Teóricos , Proteínas de Transporte de Monossacarídeos/química , Multimerização ProteicaRESUMO
Dynamic protein-DNA interactions constitute highly robust cellular machineries to fulfill cellular functions. A vast number of studies have focused on how DNA-binding proteins search for and interact with their target DNA segments and on what cellular cues can regulate protein binding, for which protein concentration is a most obvious one. In contrast, how protein unbinding could be regulated by protein concentration has evaded attention because protein unbinding from DNA is typically a unimolecular reaction and thus concentration independent. Recent single-molecule studies from multiple research groups have uncovered that protein concentration can facilitate the unbinding of DNA-bound proteins, revealing regulation of protein unbinding as another mechanistic paradigm for gene regulation. In this Account, we review these recent in vitro and in vivo single-molecule experiments that uncovered the concentration-facilitated protein unbinding by multiple types of DNA-binding proteins, including sequence-nonspecific DNA-binding proteins (e.g., nucleoid-associated proteins, NAP), sequence-specific DNA-binding proteins (e.g., metal-responsive transcription regulators CueR and ZntR), sequence-neutral single-stranded DNA-binding proteins (e.g., Replication protein A, RPA), and DNA polymerases. For the in vitro experiments, Marko's group investigated the exchange of GFP-tagged DNA-bound NAPs with nontagged NAPs in solution of increasing concentration using single-molecule magnetic-tweezers fluorescence microscopy. The faster fluorescence intensity decrease with higher nontagged NAP concentrations suggests that DNA-bound NAPs undergo faster exchange with higher free NAP concentrations. Chen's group used single-molecule fluorescence resonance energy transfer measurements to study the unbinding of CueR from its cognate oligomeric DNA. The average microscopic dwell times of DNA-bound states become shorter with increasing CueR concentrations in the surroundings, demonstrating that free CueR proteins can facilitate the unbinding of the incumbent one on DNA through either assisted dissociation or direct substitution. Greene's group studied the unbinding of RPAs from single-stranded DNA using total internal reflection fluorescence microscopy and DNA curtain techniques. The fluorescence intensity versus time traces show faster decay with higher wild-type RPA concentrations, indicating that DNA-bound RPAs can undergo a concentration-facilitated exchange when encountering excess free RPA. van Oijen's group investigated the leading/lagging-strand polymerase exchange events in the bacteriophage T7 and E. coli replication systems using a combination of single-molecule fluorescence microscopy and DNA-flow-stretching assay. The processivity was observed to have larger decrease when the concentration of the Y526F polymerase mutant increases, indicating that the unbinding of the polymerase is also concentration-dependent. Using stroboscopic imaging and single-molecule tracking, Chen's group further advanced their study into living bacterial cells. They found CueR, as well as its homologue ZntR, shows concentration-enhanced unbinding from its DNA-binding site in vivo. Mechanistic consensus has emerged from these in vitro and in vivo single-molecule studies that encompass a range of proteins with distinct biological functions. It involves multivalent contacts between protein and DNA. The multivalency enables the formation of ternary complexes as intermediates, which subsequently give rise to concentration-enhanced protein unbinding. As multivalent contacts are ubiquitous among DNA-interacting proteins, this multivalency-enabled facilitated unbinding mechanism thus provides a potentially general mechanistic paradigm in regulating protein-DNA interactions.
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DNA/química , Proteínas/químicaRESUMO
Multicomponent efflux complexes constitute a primary mechanism for Gram-negative bacteria to expel toxic molecules for survival. As these complexes traverse the periplasm and link inner and outer membranes, it remains unclear how they operate efficiently without compromising periplasmic plasticity. Combining single-molecule superresolution imaging and genetic engineering, we study in living Escherichia coli cells the tripartite efflux complex CusCBA of the resistance-nodulation-division family that is essential for bacterial resistance to drugs and toxic metals. We find that CusCBA complexes are dynamic structures and shift toward the assembled form in response to metal stress. Unexpectedly, the periplasmic adaptor protein CusB is a key metal-sensing element that drives the assembly of the efflux complex ahead of the transcription activation of the cus operon for defending against metals. This adaptor protein-mediated dynamic pump assembly allows the bacterial cell for efficient efflux upon cellular demand while still maintaining periplasmic plasticity; this could be broadly relevant to other multicomponent efflux systems.
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Cobre/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Prata/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Transporte de Íons/fisiologia , Proteínas de Membrana/genética , Proteínas de Membrana Transportadoras/genética , Periplasma/genética , Periplasma/metabolismoRESUMO
In bacteria, trigger factor (TF) is the molecular chaperone that interacts with the ribosome to assist the folding of nascent polypeptides. Studies in vitro have provided insights into the function and mechanism of TF. Much is to be elucidated, however, about how TF functions in vivo. Here, we use single-molecule tracking, in combination with genetic manipulations, to study the dynamics and function of TF in living E. coli cells. We find that TF, besides interacting with the 70S ribosome, may also bind to ribosomal subunits and form TF-polypeptide complexes that may include DnaK/DnaJ proteins. The TF-70S ribosome interactions are highly dynamic inside cells, with an average residence time of â¼0.2 s. Our results confirm that the signal recognition particle weakens TF's interaction with the 70S ribosome, and further identify that this weakening mainly results from a change in TF's binding to the 70S ribosome, rather than its unbinding. Moreover, using photoconvertible bimolecular fluorescence complementation, we selectively probe TF2 dimers in the cell and show that TF2 does not bind to the 70S ribosome but is involved in the post-translational interactions with polypeptides. These findings contribute to the fundamental understanding of molecular chaperones in assisting protein folding in living cells.
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Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Peptidilprolil Isomerase/genética , Peptidilprolil Isomerase/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Chaperonas Moleculares/metabolismo , Peptídeos/metabolismo , Peptidilprolil Isomerase/química , Ligação Proteica , Biossíntese de Proteínas , Dobramento de Proteína , Ribossomos/metabolismoRESUMO
The splitting of water photoelectrochemically into hydrogen and oxygen represents a promising technology for converting solar energy to fuel. The main challenge is to ensure that photogenerated holes efficiently oxidize water, which generally requires modification of the photoanode with an oxygen evolution catalyst (OEC) to increase the photocurrent and reduce the onset potential. However, because excess OEC material can hinder light absorption and decrease photoanode performance, its deposition needs to be carefully controlled--yet it is unclear which semiconductor surface sites give optimal improvement if targeted for OEC deposition, and whether sites catalysing water oxidation also contribute to competing charge-carrier recombination with photogenerated electrons. Surface heterogeneity exacerbates these uncertainties, especially for nanostructured photoanodes benefiting from small charge-carrier transport distances. Here we use super-resolution imaging, operated in a charge-carrier-selective manner and with a spatiotemporal resolution of approximately 30 nanometres and 15 milliseconds, to map both the electron- and hole-driven photoelectrocatalytic activities on single titanium oxide nanorods. We then map, with sub-particle resolution (about 390 nanometres), the photocurrent associated with water oxidation, and find that the most active sites for water oxidation are also the most important sites for charge-carrier recombination. Site-selective deposition of an OEC, guided by the activity maps, improves the overall performance of a given nanorod--even though more improvement in photocurrent efficiency correlates with less reduction in onset potential (and vice versa) at the sub-particle level. Moreover, the optimal catalyst deposition sites for photocurrent enhancement are the lower-activity sites, and for onset potential reduction the optimal sites are the sites with more positive onset potential, contrary to what is obtainable under typical deposition conditions. These findings allow us to suggest an activity-based strategy for rationally engineering catalyst-improved photoelectrodes, which should be widely applicable because our measurements can be performed for many different semiconductor and catalyst materials.
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Single-molecule tracking (SMT) of fluorescently tagged cytoplasmic proteins can provide valuable information on the underlying biological processes in living cells via subsequent analysis of the displacement distributions; however, the confinement effect originated from the small size of a bacterial cell skews the protein's displacement distribution and complicates the quantification of the intrinsic diffusive behaviors. Using the inverse transformation method, we convert the skewed displacement distribution (for both 2D and 3D imaging conditions) back to that in free space for systems containing one or multiple (non)interconverting Brownian diffusion states, from which we can reliably extract the number of diffusion states as well as their intrinsic diffusion coefficients and respective fractional populations. We further demonstrate a successful application to experimental SMT data of a transcription factor in living E. coli cells. This work allows a direct quantitative connection between cytoplasmic SMT data with diffusion theory for analyzing molecular diffusive behavior in live bacteria.