Single Emitter Localization

Single Emitter Localization | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Single Emitter Localization Tamoghna Bhattacharyya This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3605655/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 12 Jan, 2024 Read the published version in Journal of Fluorescence → Version 1 posted 7 You are reading this latest preprint version Abstract Understanding the relationships between molecular organization and dynamics of a complex system is very important to understand the photophysical properties of such system. This paper focuses on a novel strategy based on single molecule spectroscopy and single molecule localization microscopy to elucidate the organization, photostability and localization of a single molecule on a 2D biomembrane. Improvement of in-plane resolution of a signal in a nano-dimension within the diffraction limit has been discussed in a new way. And, how this better in-plane resolution information can be used for precise localization of a single molecule on a 2D system has also been discussed. Single molecule spectroscopy Single molecule localization microscopy Photostability Surface plasmon resonance Nanoscopy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Enhancement of photostability and quantum efficiency of a molecule has always been a priority for opto-electronics and bioelectronics.( 1 – 5 ) But, the long-term operational photostability is still at infancy level.( 6 ) The plasmonic effects is well known to improve the efficiency of light absorption and emission.( 7 – 12 ) from a decade. A metal film, ( 13 ) has been successfully used to enhance emission intensities via surface plasmon resonance. ( 14 , 15 ) Lakowicz and coworkers have shown that organic dyes are twice as stable when deposited on plasmonic silver island versus on glass.( 16 , 17 ) Kreiter et al. showed that DilC1, an organic dye is more stable of a factor of two when placed on a 20 nm flat Au surface than on an uncoated substrate.( 18 ) However, all the reported enhancements are less than a factor of ten. ( 19 – 25 ) A significant order of magnitude (about a factor of fourty) increment in stability is achieved for a fluorophore (alexa 488) coupled with a soft 2D lipid bilayer surface on top of a plasmonic film (1nm Au film). Further this surface plasmon enhanced information has been used to increase the in-plane resolution within the diffraction limit and further the precise localization of a single molecule on a 2D surface has been investigated. Results and discussions Lipid bilayer conjugated with a fluorophore on a plasmonic surface has been investigated to understand the photostability and localization of a single molecule on a 2D surface. This paper focuses on the longer photostability of a fluorophore, which has been used to develop better in-plane resolution platform to study the localization of a fluorophore on a 2D bilayer system using a hand-made total internal reflection fluorescence microscopy i.e. TIRF (see Fig. 1 a). The blinking information from each single molecule (as shown in Fig. 1 b) has further been analyzed. An optimum thickness of 20 nm that includes a lipid bilayer film and a plasmonic surface has been standardized to achieve better photostability (data not shown). The lipid bilayer serves as a spacer layer in between plasmonic surface and the fluorophore. Localized surface plasmon resonance enhancement of the electromagnetic fields of a few nanometer thick fluorophore conjugated lipid bilayer film deposited on a 1 nm Au thin film was observed. The thickness of the gold film (see Fig. S1 and Fig. S2) was optimized because the more the overlap in between the surface plasmon resonances of the plasmonic film with the emission of Alexa 488, the more the photostability can be achieved. A series of thickness of Au film was investigated (Fig. 1 c) and found that 1 nm Au film results better photostability and that is because of maximum overlap in between the surface plasmon resonances of the plasmonic film with the emission of Alexa 488. Experiment was further carried out for longer time to understand the plasmonic effect on photostability in a longer time period (Fig. 1 c). Even after 45 minutes the intensity of Alexa 488 covalently attached with lipid bilayer surface deposited on top of a 1 nm gold surface didn’t decrease (Fig. 1 c, red color) much whereas it was hardly be seen on just top of a glass surface (Fig. 1 c, black color). The dependence of thickness of the plasmonic surface was investigated, and it was observed that the photostability decreased with increase in thickness of the plasmonic film (results are shown in Fig. 1 c). The thicker plasmonic film (2 nm Au film and 4 nm Au film) quenches the fluorescence intensity of the fluorophore. It was also observed that the photostability decreased asymptotically with increase of the thickness of Au film (Fig. 1 c, green color and Fig. 1 c, purple color). Almost a factor of fourty in photostability was achieved using a 1nm gold film. To further confirm the importance of the plasmon peak position in achieving photo stability enhancement, alexa 488 embedded bilayer was deposited onto a glass substrate on top of a 1 nm thick film of copper (Cu). The plasmon of Cu absorbs at lower energy and does not spectrally overlap well with the emission of alexa 488 and resulted less photostability (Fig. 1 c, blue color). These results confirm that good spectral overlap of the molecular emission with the plasmon absorption is required to achieve enhanced photo-stability. In addition to the photo-stability enhancement brought about through plasmonic interactions (Fig. 2 a), single molecules of alexa 488 show significantly reduced fluorescence intermittency on the Au substrate (Fig. 2 b). The ‘on-off’ emission switching that occurs on the timescale of seconds is because of the reversible formation of a polaron due to energy release of an electron to aerial oxygen. The fluorescence lifetimes were measured (Fig. 2 c). Alexa 488 single molecules exhibit single exponential decay kinetics on glass (lifetime 3.8 ns). On 2nm Au, the decay rate is similar to the glass but on 1nm Au the decay rate is much faster (lifetime 1.2 ns). The observed decrease in polaron formation and less photo-degradation on 1 nm Au film are both consistent with a reduction in the excited state lifetimes due to plasmonic interactions. Further, second order co-relation measurements were carried out to confirm the single molecule study. Organic molecules pumped at low excitation rates, means that there is one excitation and emission event within a single laser pulse. This relaxation time is governed by the triplet lifetime. It is also possible to observe two emission events within a single laser pulse if; the ground state is re-populated due to rapid radiative decay. g2 (τ) showed the anti-bunching behavior which demonstrates that the predominant emitters are single molecules rather than aggregates (see Fig. 2 d). We performed the experiment from bulk and gradually decreasing the concentration of the fluorophore to achieve single molecule. We observed bunching at bulk and also for the aggregates (the black and purple color in Fig. 2 d confirmed it) and finally we got anti-bunching (as shown in blue color in Fig. 2 d). The value was found to be 0.3. The rigidity of the bilayer (see Fig. S3-Fig. S5) was further changed so that we can track the lateral translocation of a single fluorescent molecule. The molecular positions of the molecule were extracted from the videos captured at single molecule level. Under ideal conditions, the average intensity produced by a single emitter is proportional to the cross-section of the microscope’s point spread function (PSF). I(x^',y^' ) = H(x,y,z,N s ,N b ) = H(θ); Where x’, y’ the positions in the image plane when the emitter is at position x, y, z in the sample space, where θ represents these position and photon parameters; Ns is the total signal photons and N b is the constant background per unit area. The actual image from the detector is subject to noise and binned into pixels, producing a signal nk at each of the pixels wk within the region of interest (ROI). Therefore, the position estimator can be defined by x^''=(∑n k .w k )⁄(∑n k ) The imaging model H (θ) produces an expected value of the image, I (x’, y’; θ), and the best guess of θ is found by optimizing I to match the observed PSF. LS was one of the first relatively unbiased localization algorithms used ( 26 , 27 ) and is familiar from its ubiquitous application to scientific regression problems and curve fitting. The scoring method of LS, ( 27 , 28 ) is to minimize the square error between the PSF model u (θ) and the observed data can best be described by S=∑[ n k -u k (θ)] 2 When molecules emit light isotopically, without net polarization, the scalar approximation allows for a comprehensive treatment of the imaging system The image of an ideal, in-focus emitter at the coverslip can be best described by I(x^',y^' ) = C[(J(kNA ρ ))⁄(kNA ρ ] 2 ) where J being the first-order Bessel function of the first kind, k the wavenumber 2π/λ, ρ the distance from the point source in the image plane and C a constant for a given number of total detected photons. As the PSF’s intensity is concentrated in the center, therefore, a Gaussian function is a tractable and reasonably accurate approximation, and it is extremely common to fit single-molecule data with a symmetric Gaussian plus a constant background with the width σ the size of the spot arising from the diffraction limit ( 27 , 28 ) I(x^',y^' ) = N s /2πσ 2 exp (-ρ 2 /2σ 2 ) + N b Based on this model a position of the conjugated fluorophore was estimated close to that of the true position that avoids the bias of the simpler estimators. But the essential question is how precise an individual localization is. To address this, we can either simulate or measure the spread of position estimates obtained from the same molecule emitting over many frames. Repeated least-squares localizations on simulated data give a range of estimates with standard deviation, or in other words, the localization precision can be achieved. But again, the localization precision is dependent on the estimator also. Therefore, the correction for localization precision ( 27 , 28 ) which is best described by the proposed equation as described below. σ 2 =(σ i 2 + a 2 /12)/N s (16/9 + 8πNb(σ i 2 + a 2 /12)/(N s .a 2 ); Where standard deviation σi detected with pixels of area a2 and gives the correct result with a low background and high intensity i.e., due to presence of surface plasmon, and concisely predicts the precise localization. Based on this model, a strategy has been proposed to understand the lateral localization of single molecule on a 2D surface (scheme has been provided in Fig. 3 a). Histograms of standard deviations of localizations from single molecules in x and y has been shown in Fig. 3 b. It's clear seen that when the labeled lipid concentration is lower the average mean free path between labeled lipids increases (Fig. 4a), and that's why the distribution of pixel intensity is getting decreased (Fig. 4b). In other words, bilayers with higher lipid concentration are more constrained, and therefore, the diffusion is rather more restricted (as shown in Distance vs. Time graph in Fig. 4c and Fig. 4d) and that's why the distribution of average intensity doesn't change significantly within the ROI. Based on this information further simulation has been carried out. A single Alexa 488 molecule embedded on a lipid bilayer deposited on a 1 nm gold surface was horizontally moved to test the ability to measure the thickness of the plasmonic surface precisely with the step sizes of the heterostructure. Fluorescent images were collected with 0.5-s integration time. Several individual spots (optimized with the maximum pixel intensity as shown in Fig. 5 a), corresponding to different concentration of Alexa 488 conjugated bilayer and approximately 5,000 to 10,000 photons per spot per image were collected, enabling us to locate the center to within the range of 20 nm to 80 nm typically, and, for brighter spots, 40 nm (see Fig. 5 b). Conclusion The photostability of Alexa 488 was increased significantly by depositing it onto ultra-thin 1 nm gold films that are optimized to achieve maximal enhancement of the stability with minimal emission quenching. A model has been proposed to understand the localization of a single fluorophore embedded on a 2D surface based on single molecule investigation. The specificity and sensitivity of single molecule fluorescence in presence of a plasmonic surface, combined with the nanometer spatial localization and enhanced photostability of a single fluorophore on a 2D surface is presented here. Declarations Acknowledgements Funding: The author acknowledges support from National Science Federation for the work. Conflict of interest: The author declares no conflict of interest associated with this paper. References Tsang DPK, Matsushima T, Adachi C (2016) Operational Stability Enhancement in Organic Light-Emitting Diodes with Ultrathin Liq Interlayers. Sci. Rep. 6: 22463. O’Carroll DM, Hofmann CE, Atwater HA (2010) Conjugated Polymer/Metal Nanowire Heterostructure Plasmonic Antennas. Adv. Mater. 22:1223–1227. Carter C, Shen Z, Zhu K, Gwynne K, O’Carroll DM (2018) Photoluminescence Stability of Blue Organic Phosphorescent Materials on Plasmonic Silver Nanostructure Architectures. In Organic Photonic 73 Materials and Devices XX; International Society for Optics and Photonics 10529: 1052907. Shen Z, O’Carroll DM (2015) Metal Films: Nano porous Silver Thin Films: Multifunctional Platforms for Influencing Chain Morphology and Optical Properties of Conjugated Polymers Adv. Funct. Mater. 25:3443–3443. Moynihan S, Iacopino D, O’Carroll D, Doyle H, Tanner DA, Redmond G (2007) Emission Colour Tuning in Semiconducting Polymer Nanotubes by Energy Transfer to Organo- Lanthanide Dopants. Adv. Mater. 19: 2474–2479. Scholz S, Kondakov D, Lüssem B, Leo K (2015) Degradation Mechanisms and Reactions in Organic Light-Emitting Devices. Chem. Rev. 115: 8449–8503. Gu X, Qiu T, Zhang W, Chu PK (2011) Light-Emitting Diodes Enhanced by Localized Surface Plasmon Resonance. Nanoscale Res. Lett. 6: 199. Xiao Y, Yang JP, Cheng PP, Zhu JJ, Xu ZQ, Deng YH, Lee ST, Li YQ, Tang JX (2012) Surface Plasmon-Enhanced Electroluminescence in Organic Light-Emitting Diodes Incorporating Au Nanoparticles. Appl. Phys. Lett. 100: 013308. Darvill D, Centeno A, Xie F (2013) Plasmonic Fluorescence Enhancement by Metal Nanostructures: Shaping the Future of Bionanotechnology. Phys. Chem. Chem. Phys. 15: 15709–15726. Liang Z, Sun J, Jiang Y, Jiang L, Chen X (2014) Plasmonic Enhanced Optoelectronic Devices. Plasmonics 9: 859–866. Dong J, Zhang Z, Zheng H, Sun M (2015) Recent Progress on Plasmon-Enhanced Fluorescence. Nanophononics 4: 472–490. Malicka J, Gryczynski I, Fang J, Kusba J, Lakowicz JR (2002) Photostability of Cy3 and Cy5-Labeled DNA in the Presence of Metallic Silver Particles. J. Fluoresc. 12: 439–447. Geddes CD, Cao H, Lakowicz JR (2003) Enhanced Photostability of ICG in Close Proximity to Gold Colloids. Spectrochim. Acta. A. Mol. Biomol. Spectrosc. 59: 2611–2617. Vasilev K, Stefani FD, Jacobsen V, Knoll W, Kreiter M (2004) Reduced Photobleaching of Chromophores Close to a Metal Surface. J. Chem. Phys. 120: 6701–6704. Park JH, Lim YT, Park OO, Kim YC (2003) Enhancement of Photostability in Blue-Light-Emitting Polymers Doped with Gold Nanoparticles. Macromol. Rapid Commun. 24: 331–334. Fu Y, Zhang J, Lakowicz JR (2008) Reduced Blinking and Long-Lasting Fluorescence of Single Fluorophores Coupling to Silver Nanoparticles. Langmuir 24: 3429– 3433. Luchowski R, Shtoyko T, Apicella E, Sarkar P, Akopova I, Raut S, Fudala R, Borejdo J, Gryczynski Z, Gryczynski I (2011) Fractal-Like Silver Aggregates Enhance the Brightness and Stability of Single-Molecule Fluorescence. Appl. Spectrosc. 65: 174–180. Cang H, Liu Y, Wang Y, Yin X, Zhang X (2013) Giant Suppression of Photobleaching for Single Molecule Detection via the Purcell Effect. Nano Lett. 13: 5949–5953. Pellegrotti JV, Acuna GP, Puchkova A, Holzmeister P, Gietl A, Lalkens B, Stefani FD, Tinnefeld P (2014) Controlled Reduction of Photobleaching in DNA Origami–Gold Nanoparticle Hybrids. Nano Lett. 14: 2831–2836. Donehue JE, Wertz E, Talicska CN, Biteen JS (2014) Plasmon-Enhanced Brightness and Photostability from Single Fluorescent Proteins Coupled to Gold Nanorods. J. Phys. Chem. C 118: 15027–15035. Peters VN, Tumkur TU, Zhu G, Noginov MA (2015) Control of a Chemical Reaction with Nonlocal Dielectric Environments. Sci. Rep. 5: 14620. Anderson CM, Georgiou GN, Morrison IE, Stevenson GV, Cherry RJCJ (1992) Cell. Sci. 101: 415– 425 Cheezum MK, Walker WF, Guilford WH (2001) Biophys. J. 81: 2378– 2388 Gibson SF, Lanni F (1991) Experimental test of an analytical model of aberration in an oil-immersion objective lens used in three-dimensional light microscopy J. Opt. Soc. Am. A 8: 1601– 1613 Abraham AV, Ram S, Chao J, Ward ES, Ober RJ (2009) Quantitative study of single molecule location estimation techniques Opt. Express 17: 23352– 23373. Zhang B, Zerubia J, Olivo-Marin J (2007) Gaussian approximations of fluorescence microscope point-spread function models Appl. Opt. 46: 1819–1829. Mortensen KI, Churchman LS, Spudich JA, Flyvbjerg H (2010) Optimized localization analysis for single-molecule tracking and super-resolution microscopy Nat. Methods 7: 377– 381. Thompson RE, Larson DR, Webb WW (2002) Precise Nanometer Localization Analysis for Individual Fluorescent Probes Biophys. J. 82: 2775– 2783. Additional Declarations No competing interests reported. Supplementary Files Supplementalmaterials.docx Supplemental materials: More data both at bulk and single molecule level and Materials & methods have been provided which are available free of charge on the Springer website. GA.png Graphical Abstract Localization study at single molecule level Cite Share Download PDF Status: Published Journal Publication published 12 Jan, 2024 Read the published version in Journal of Fluorescence → Version 1 posted Editorial decision: Revision requested 18 Dec, 2023 Reviews received at journal 15 Dec, 2023 Reviewers agreed at journal 07 Dec, 2023 Reviewers invited by journal 21 Nov, 2023 Submission checks completed at journal 14 Nov, 2023 Editor assigned by journal 14 Nov, 2023 First submitted to journal 13 Nov, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3605655","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":249156125,"identity":"23985314-0e5b-4a99-8e7a-e2f4d06be1a3","order_by":0,"name":"Tamoghna Bhattacharyya","email":"data:image/png;base64,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","orcid":"","institution":"Carnegie Mellon University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Tamoghna","middleName":"","lastName":"Bhattacharyya","suffix":""}],"badges":[],"createdAt":"2023-11-13 14:59:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3605655/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3605655/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10895-023-03580-x","type":"published","date":"2024-01-12T15:01:31+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":46569653,"identity":"fad26086-d46a-4202-8a70-d6c605ca756f","added_by":"auto","created_at":"2023-11-16 15:50:11","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":125902,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Schematic of Total Internal Reflection Microscopy (TIRF) setup used for this experiment; (b) Blinking image captured from TRF measurement. Convergence of iterative localizations for molecules within the activation area; (c) Photostability study of a single molecule: alexa 488 on a glass surface (black), alexa 488 on a 4 nm gold film (purple), alexa 488 embedded on lipid bilayer on a 2 nm gold film (green), alexa 488 embedded on lipid bilayer on a 1 nm gold film (red) and, alexa 488 embedded on lipid bilayer on a 1 nm copper film (blue)\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-3605655/v1/cfdc0b0905500091a595771d.png"},{"id":46569654,"identity":"d0efd505-14c6-4a21-a425-3c46dba1394a","added_by":"auto","created_at":"2023-11-16 15:50:11","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":98412,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Photostability study of a single alexa 488 molecule (Black: on glass, Blue: on 1 nm Au film) (b) Fluorescence intermittence study; (c) Lifetime of alexa 488 on glass (red); on 1 nm Cu film (green) and 1 nm Au film (black); and (d) Second order co-relation (g2) measurements of a single alexa 488 molecules.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-3605655/v1/ff5323bdd45f3715abab5917.png"},{"id":46569657,"identity":"86c2a263-5829-42f8-a3f4-212bdd7b0261","added_by":"auto","created_at":"2023-11-16 15:50:11","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":58116,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Scheme of localization study; (b) Histograms of standard deviation of localizations from single molecules in x and y\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-3605655/v1/1b34190caf0ce596146d68bc.png"},{"id":46569655,"identity":"f51550bd-6d00-4a3c-9b64-1b3c64c4fd72","added_by":"auto","created_at":"2023-11-16 15:50:11","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":116331,"visible":true,"origin":"","legend":"\u003cp\u003e(a), (b) Average mean free path study conjugated fluorophore; (c), (d) Diffusion study of a single emitter\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-3605655/v1/89b044b5459a4db151f4a91e.png"},{"id":46569656,"identity":"43bcf607-f3c1-4cdf-a6f6-64452e237fc3","added_by":"auto","created_at":"2023-11-16 15:50:11","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":50504,"visible":true,"origin":"","legend":"\u003cp\u003eDependence of plasmonic stability with the step size of the heterostructure\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-3605655/v1/28d65ae62094c792a5e1e769.png"},{"id":49628764,"identity":"84c8ff17-4169-49dc-9c39-e765a481e5b0","added_by":"auto","created_at":"2024-01-15 15:09:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":584219,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3605655/v1/74aae6fd-65f8-45e3-995d-f492e0ae99aa.pdf"},{"id":46569658,"identity":"9e2185a5-4ac9-4c64-a8fc-ccea3da07e8e","added_by":"auto","created_at":"2023-11-16 15:50:11","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":403125,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplemental materials: \u003c/strong\u003eMore data both at bulk and single molecule level and Materials \u0026amp; methods have been provided which are available free of charge on the Springer website.\u003c/p\u003e","description":"","filename":"Supplementalmaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-3605655/v1/cce475a5df44728c74fe7043.docx"},{"id":46569660,"identity":"a9b88b79-ab51-48fe-b2af-735de563c49d","added_by":"auto","created_at":"2023-11-16 15:50:11","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":32232,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphical Abstract\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLocalization study at single molecule level\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"GA.png","url":"https://assets-eu.researchsquare.com/files/rs-3605655/v1/6fe24f6e04f96faf96b99227.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Single Emitter Localization","fulltext":[{"header":"Introduction","content":"\u003cp\u003eEnhancement of photostability and quantum efficiency of a molecule has always been a priority for opto-electronics and bioelectronics.(\u003cspan additionalcitationids=\"CR2 CR3 CR4\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e) But, the long-term operational photostability is still at infancy level.(\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e) The plasmonic effects is well known to improve the efficiency of light absorption and emission.(\u003cspan additionalcitationids=\"CR8 CR9 CR10 CR11\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e) from a decade. A metal film, (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e) has been successfully used to enhance emission intensities via surface plasmon resonance. (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e) Lakowicz and coworkers have shown that organic dyes are twice as stable when deposited on plasmonic silver island versus on glass.(\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e) Kreiter et al. showed that DilC1, an organic dye is more stable of a factor of two when placed on a 20 nm flat Au surface than on an uncoated substrate.(\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e) However, all the reported enhancements are less than a factor of ten. (\u003cspan additionalcitationids=\"CR20 CR21 CR22 CR23 CR24\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e) A significant order of magnitude (about a factor of fourty) increment in stability is achieved for a fluorophore (alexa 488) coupled with a soft 2D lipid bilayer surface on top of a plasmonic film (1nm Au film). Further this surface plasmon enhanced information has been used to increase the in-plane resolution within the diffraction limit and further the precise localization of a single molecule on a 2D surface has been investigated.\u003c/p\u003e"},{"header":"Results and discussions","content":"\u003cp\u003eLipid bilayer conjugated with a fluorophore on a plasmonic surface has been investigated to understand the photostability and localization of a single molecule on a 2D surface. This paper focuses on the longer photostability of a fluorophore, which has been used to develop better in-plane resolution platform to study the localization of a fluorophore on a 2D bilayer system using a hand-made total internal reflection fluorescence microscopy i.e. TIRF (see Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea). The blinking information from each single molecule (as shown in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eb) has further been analyzed. An optimum thickness of 20 nm that includes a lipid bilayer film and a plasmonic surface has been standardized to achieve better photostability (data not shown). The lipid bilayer serves as a spacer layer in between plasmonic surface and the fluorophore.\u003c/p\u003e\n\u003cp\u003eLocalized surface plasmon resonance enhancement of the electromagnetic fields of a few nanometer thick fluorophore conjugated lipid bilayer film deposited on a 1 nm Au thin film was observed. The thickness of the gold film (see Fig. \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e and Fig. S2) was optimized because the more the overlap in between the surface plasmon resonances of the plasmonic film with the emission of Alexa 488, the more the photostability can be achieved. A series of thickness of Au film was investigated (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ec) and found that 1 nm Au film results better photostability and that is because of maximum overlap in between the surface plasmon resonances of the plasmonic film with the emission of Alexa 488. Experiment was further carried out for longer time to understand the plasmonic effect on photostability in a longer time period (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ec). Even after 45 minutes the intensity of Alexa 488 covalently attached with lipid bilayer surface deposited on top of a 1 nm gold surface didn\u0026rsquo;t decrease (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ec, red color) much whereas it was hardly be seen on just top of a glass surface (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ec, black color). The dependence of thickness of the plasmonic surface was investigated, and it was observed that the photostability decreased with increase in thickness of the plasmonic film (results are shown in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ec). The thicker plasmonic film (2 nm Au film and 4 nm Au film) quenches the fluorescence intensity of the fluorophore. It was also observed that the photostability decreased asymptotically with increase of the thickness of Au film (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ec, green color and Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ec, purple color). Almost a factor of fourty in photostability was achieved using a 1nm gold film. To further confirm the importance of the plasmon peak position in achieving photo stability enhancement, alexa 488 embedded bilayer was deposited onto a glass substrate on top of a 1 nm thick film of copper (Cu). The plasmon of Cu absorbs at lower energy and does not spectrally overlap well with the emission of alexa 488 and resulted less photostability (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ec, blue color). These results confirm that good spectral overlap of the molecular emission with the plasmon absorption is required to achieve enhanced photo-stability. In addition to the photo-stability enhancement brought about through plasmonic interactions (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea), single molecules of alexa 488 show significantly reduced fluorescence intermittency on the Au substrate (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eb). The \u0026lsquo;on-off\u0026rsquo; emission switching that occurs on the timescale of seconds is because of the reversible formation of a polaron due to energy release of an electron to aerial oxygen. The fluorescence lifetimes were measured (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ec). Alexa 488 single molecules exhibit single exponential decay kinetics on glass (lifetime 3.8 ns). On 2nm Au, the decay rate is similar to the glass but on 1nm Au the decay rate is much faster (lifetime 1.2 ns). The observed decrease in polaron formation and less photo-degradation on 1 nm Au film are both consistent with a reduction in the excited state lifetimes due to plasmonic interactions.\u003c/p\u003e\n\u003cp\u003eFurther, second order co-relation measurements were carried out to confirm the single molecule study. Organic molecules pumped at low excitation rates, means that there is one excitation and emission event within a single laser pulse. This relaxation time is governed by the triplet lifetime. It is also possible to observe two emission events within a single laser pulse if; the ground state is re-populated due to rapid radiative decay. g2 (\u0026tau;) showed the anti-bunching behavior which demonstrates that the predominant emitters are single molecules rather than aggregates (see Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ed). We performed the experiment from bulk and gradually decreasing the concentration of the fluorophore to achieve single molecule. We observed bunching at bulk and also for the aggregates (the black and purple color in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ed confirmed it) and finally we got anti-bunching (as shown in blue color in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ed). The value was found to be 0.3.\u003c/p\u003e\n\u003cp\u003eThe rigidity of the bilayer (see Fig. S3-Fig. S5) was further changed so that we can track the lateral translocation of a single fluorescent molecule. The molecular positions of the molecule were extracted from the videos captured at single molecule level. Under ideal conditions, the average intensity produced by a single emitter is proportional to the cross-section of the microscope\u0026rsquo;s point spread function (PSF).\u003c/p\u003e\n\u003cp\u003eI(x^\u0026apos;,y^\u0026apos; )\u0026thinsp;=\u0026thinsp;H(x,y,z,N\u003csub\u003es\u003c/sub\u003e,N\u003csub\u003eb\u003c/sub\u003e)\u0026thinsp;=\u0026thinsp;H(\u0026theta;); Where x\u0026rsquo;, y\u0026rsquo; the positions in the image plane when the emitter is at position x, y, z in the sample space, where \u0026theta; represents these position and photon parameters; Ns is the total signal photons and N\u003csub\u003eb\u003c/sub\u003e is the constant background per unit area. The actual image from the detector is subject to noise and binned into pixels, producing a signal nk at each of the pixels wk within the region of interest (ROI).\u003c/p\u003e\n\u003cp\u003eTherefore, the position estimator can be defined by\u003c/p\u003e\n\u003cp\u003ex^\u0026apos;\u0026apos;=(\u0026sum;n\u003csub\u003ek\u003c/sub\u003e.w\u003csub\u003ek\u003c/sub\u003e )\u0026frasl;(\u0026sum;n\u003csub\u003ek\u003c/sub\u003e)\u003c/p\u003e\n\u003cp\u003eThe imaging model H (\u0026theta;) produces an expected value of the image, I (x\u0026rsquo;, y\u0026rsquo;; \u0026theta;), and the best guess of \u0026theta; is found by optimizing I to match the observed PSF. LS was one of the first relatively unbiased localization algorithms used (\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e) and is familiar from its ubiquitous application to scientific regression problems and curve fitting. The scoring method of LS, (\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e) is to minimize the square error between the PSF model u (\u0026theta;) and the observed data can best be described by\u003c/p\u003e\n\u003cp\u003eS=\u0026sum;[ n\u003csub\u003ek\u003c/sub\u003e-u\u003csub\u003ek\u003c/sub\u003e(\u0026theta;)]\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eWhen molecules emit light isotopically, without net polarization, the scalar approximation allows for a comprehensive treatment of the imaging system The image of an ideal, in-focus emitter at the coverslip can be best described by\u003c/p\u003e\n\u003cp\u003eI(x^\u0026apos;,y^\u0026apos; )\u0026thinsp;=\u0026thinsp;C[(J(kNA\u003csub\u003e\u0026rho;\u003c/sub\u003e))\u0026frasl;(kNA\u003csub\u003e\u0026rho;\u003c/sub\u003e]\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e\n\u003cp\u003ewhere J being the first-order Bessel function of the first kind, k the wavenumber 2\u0026pi;/\u0026lambda;, \u0026rho; the distance from the point source in the image plane and C a constant for a given number of total detected photons. As the PSF\u0026rsquo;s intensity is concentrated in the center, therefore, a Gaussian function is a tractable and reasonably accurate approximation, and it is extremely common to fit single-molecule data with a symmetric Gaussian plus a constant background with the width \u0026sigma; the size of the spot arising from the diffraction limit (\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e)\u003c/p\u003e\n\u003cp\u003eI(x^\u0026apos;,y^\u0026apos; )\u0026thinsp;=\u0026thinsp;N\u003csub\u003es\u003c/sub\u003e/2\u0026pi;\u0026sigma;\u003csup\u003e2\u003c/sup\u003e exp (-\u0026rho;\u003csup\u003e2\u003c/sup\u003e/2\u0026sigma;\u003csup\u003e2\u003c/sup\u003e)\u0026thinsp;+\u0026thinsp;N\u003csub\u003eb\u003c/sub\u003e\u003c/p\u003e\n\u003cp\u003eBased on this model a position of the conjugated fluorophore was estimated close to that of the true position that avoids the bias of the simpler estimators. But the essential question is how precise an individual localization is. To address this, we can either simulate or measure the spread of position estimates obtained from the same molecule emitting over many frames. Repeated least-squares localizations on simulated data give a range of estimates with standard deviation, or in other words, the localization precision can be achieved. But again, the localization precision is dependent on the estimator also. Therefore, the correction for localization precision (\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e) which is best described by the proposed equation as described below.\u003c/p\u003e\n\u003cp\u003e\u0026sigma;\u003csup\u003e2\u003c/sup\u003e=(\u0026sigma;\u003csub\u003ei\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;+\u0026thinsp;a\u003csup\u003e2\u003c/sup\u003e/12)/N\u003csub\u003es\u003c/sub\u003e(16/9\u0026thinsp;+\u0026thinsp;8\u0026pi;Nb(\u0026sigma;\u003csub\u003ei\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;+\u0026thinsp;a\u003csup\u003e2\u003c/sup\u003e/12)/(N\u003csub\u003es\u003c/sub\u003e.a\u003csup\u003e2\u003c/sup\u003e); Where standard deviation \u0026sigma;i detected with pixels of area a2 and gives the correct result with a low background and high intensity i.e., due to presence of surface plasmon, and concisely predicts the precise localization. Based on this model, a strategy has been proposed to understand the lateral localization of single molecule on a 2D surface (scheme has been provided in Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea). Histograms of standard deviations of localizations from single molecules in x and y has been shown in Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eb.\u003c/p\u003e\n\u003cp\u003eIt\u0026apos;s clear seen that when the labeled lipid concentration is lower the average mean free path between labeled lipids increases (Fig. 4a), and that\u0026apos;s why the distribution of pixel intensity is getting decreased (Fig. 4b). In other words, bilayers with higher lipid concentration are more constrained, and therefore, the diffusion is rather more restricted (as shown in Distance vs. Time graph in Fig. 4c and Fig. 4d) and that\u0026apos;s why the distribution of average intensity doesn\u0026apos;t change significantly within the ROI. Based on this information further simulation has been carried out.\u003c/p\u003e\n\u003cp\u003eA single Alexa 488 molecule embedded on a lipid bilayer deposited on a 1 nm gold surface was horizontally moved to test the ability to measure the thickness of the plasmonic surface precisely with the step sizes of the heterostructure. Fluorescent images were collected with 0.5-s integration time. Several individual spots (optimized with the maximum pixel intensity as shown in Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea), corresponding to different concentration of Alexa 488 conjugated bilayer and approximately 5,000 to 10,000 photons per spot per image were collected, enabling us to locate the center to within the range of 20 nm to 80 nm typically, and, for brighter spots, 40 nm (see Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eb).\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe photostability of Alexa 488 was increased significantly by depositing it onto ultra-thin 1 nm gold films that are optimized to achieve maximal enhancement of the stability with minimal emission quenching. A model has been proposed to understand the localization of a single fluorophore embedded on a 2D surface based on single molecule investigation. The specificity and sensitivity of single molecule fluorescence in presence of a plasmonic surface, combined with the nanometer spatial localization and enhanced photostability of a single fluorophore on a 2D surface is presented here.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e The author acknowledges support from National Science Federation for the work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest:\u0026nbsp;\u003c/strong\u003eThe author declares no conflict of interest associated with this paper.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eTsang DPK, Matsushima T, Adachi C (2016) Operational Stability Enhancement in Organic Light-Emitting Diodes with Ultrathin Liq Interlayers. Sci. Rep. 6: 22463.\u003c/li\u003e\n \u003cli\u003eO\u0026rsquo;Carroll DM, Hofmann CE, Atwater HA (2010) Conjugated Polymer/Metal Nanowire Heterostructure Plasmonic Antennas. Adv. Mater. 22:1223\u0026ndash;1227.\u003c/li\u003e\n \u003cli\u003eCarter C, Shen Z, Zhu K, Gwynne K, O\u0026rsquo;Carroll DM (2018) Photoluminescence Stability of Blue Organic Phosphorescent Materials on Plasmonic Silver Nanostructure Architectures. In Organic Photonic 73 Materials and Devices XX; International Society for Optics and Photonics 10529: 1052907.\u003c/li\u003e\n \u003cli\u003eShen Z, O\u0026rsquo;Carroll DM (2015) Metal Films: Nano porous Silver Thin Films: Multifunctional Platforms for Influencing Chain Morphology and Optical Properties of Conjugated Polymers Adv. Funct. Mater. 25:3443\u0026ndash;3443.\u003c/li\u003e\n \u003cli\u003eMoynihan S, Iacopino D, O\u0026rsquo;Carroll D, Doyle H, Tanner DA, Redmond G (2007) Emission Colour Tuning in Semiconducting Polymer Nanotubes by Energy Transfer to Organo- Lanthanide Dopants. Adv. Mater. 19: 2474\u0026ndash;2479.\u003c/li\u003e\n \u003cli\u003eScholz S, Kondakov D, L\u0026uuml;ssem B, Leo K (2015) Degradation Mechanisms and Reactions in Organic Light-Emitting Devices. Chem. Rev. 115: 8449\u0026ndash;8503.\u003c/li\u003e\n \u003cli\u003eGu X, Qiu T, Zhang W, Chu PK (2011) Light-Emitting Diodes Enhanced by Localized Surface Plasmon Resonance. Nanoscale Res. Lett. 6: 199.\u003c/li\u003e\n \u003cli\u003eXiao Y, Yang JP, Cheng PP, Zhu JJ, Xu ZQ, Deng YH, Lee ST, Li YQ, Tang JX (2012) Surface Plasmon-Enhanced Electroluminescence in Organic Light-Emitting Diodes Incorporating Au Nanoparticles. Appl. Phys. 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Spectrosc. 59: 2611\u0026ndash;2617.\u003c/li\u003e\n \u003cli\u003eVasilev K, Stefani FD, Jacobsen V, Knoll W, Kreiter M (2004) Reduced Photobleaching of Chromophores Close to a Metal Surface. J. Chem. Phys. 120: 6701\u0026ndash;6704.\u003c/li\u003e\n \u003cli\u003ePark JH, Lim YT, Park OO, Kim YC (2003) Enhancement of Photostability in Blue-Light-Emitting Polymers Doped with Gold Nanoparticles. Macromol. Rapid Commun. 24: 331\u0026ndash;334.\u003c/li\u003e\n \u003cli\u003eFu Y, Zhang J, Lakowicz JR (2008) Reduced Blinking and Long-Lasting Fluorescence of Single Fluorophores Coupling to Silver Nanoparticles. Langmuir 24: 3429\u0026ndash; 3433.\u003c/li\u003e\n \u003cli\u003eLuchowski R, Shtoyko T, Apicella E, Sarkar P, Akopova I, Raut S, Fudala R, Borejdo J, Gryczynski Z, Gryczynski I (2011) Fractal-Like Silver Aggregates Enhance the Brightness and Stability of Single-Molecule Fluorescence. Appl. 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Rep. 5: 14620.\u003c/li\u003e\n \u003cli\u003eAnderson CM, Georgiou GN, Morrison IE, Stevenson GV, Cherry RJCJ (1992) Cell. Sci. 101: 415\u0026ndash; 425\u003c/li\u003e\n \u003cli\u003eCheezum MK, Walker WF, Guilford WH (2001) Biophys. J. 81: 2378\u0026ndash; 2388\u003c/li\u003e\n \u003cli\u003eGibson SF, Lanni F (1991) Experimental test of an analytical model of aberration in an oil-immersion objective lens used in three-dimensional light microscopy J. Opt. Soc. Am. A 8: 1601\u0026ndash; 1613\u003c/li\u003e\n \u003cli\u003eAbraham AV, Ram S, Chao J, Ward ES, Ober RJ (2009) Quantitative study of single molecule location estimation techniques Opt. Express 17: 23352\u0026ndash; 23373.\u003c/li\u003e\n \u003cli\u003eZhang B, Zerubia J, Olivo-Marin J (2007) Gaussian approximations of fluorescence microscope point-spread function models Appl. Opt. 46: 1819\u0026ndash;1829.\u003c/li\u003e\n \u003cli\u003eMortensen KI, Churchman LS, Spudich JA, Flyvbjerg H (2010) Optimized localization analysis for single-molecule tracking and super-resolution microscopy Nat. Methods 7: 377\u0026ndash; 381.\u003c/li\u003e\n \u003cli\u003eThompson RE, Larson DR, Webb WW (2002) Precise Nanometer Localization Analysis for Individual Fluorescent Probes Biophys. J. 82: 2775\u0026ndash; 2783.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-fluorescence","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jofl","sideBox":"Learn more about [Journal of Fluorescence](https://www.springer.com/journal/10895)","snPcode":"10895","submissionUrl":"https://submission.nature.com/new-submission/10895/3","title":"Journal of Fluorescence","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Single molecule spectroscopy, Single molecule localization microscopy, Photostability, Surface plasmon resonance, Nanoscopy ","lastPublishedDoi":"10.21203/rs.3.rs-3605655/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3605655/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eUnderstanding the relationships between molecular organization and dynamics of a complex system is very important to understand the photophysical properties of such system. This paper focuses on a novel strategy based on single molecule spectroscopy and single molecule localization microscopy to elucidate the organization, photostability and localization of a single molecule on a 2D biomembrane. Improvement of in-plane resolution of a signal in a nano-dimension within the diffraction limit has been discussed in a new way. And, how this better in-plane resolution information can be used for precise localization of a single molecule on a 2D system has also been discussed.\u003c/p\u003e","manuscriptTitle":"Single Emitter Localization","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-11-16 15:50:06","doi":"10.21203/rs.3.rs-3605655/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2023-12-18T12:01:43+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2023-12-15T19:23:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"b69b30b6-24a8-447d-a518-cd0fd1e547b2","date":"2023-12-08T00:17:05+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2023-11-21T19:27:11+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2023-11-14T08:11:24+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-11-14T08:11:24+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Fluorescence","date":"2023-11-13T14:52:23+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-fluorescence","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jofl","sideBox":"Learn more about [Journal of Fluorescence](https://www.springer.com/journal/10895)","snPcode":"10895","submissionUrl":"https://submission.nature.com/new-submission/10895/3","title":"Journal of Fluorescence","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"805180ce-241e-4d4e-a512-81f09f9da75d","owner":[],"postedDate":"November 16th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-01-15T15:06:28+00:00","versionOfRecord":{"articleIdentity":"rs-3605655","link":"https://doi.org/10.1007/s10895-023-03580-x","journal":{"identity":"journal-of-fluorescence","isVorOnly":false,"title":"Journal of Fluorescence"},"publishedOn":"2024-01-12 15:01:31","publishedOnDateReadable":"January 12th, 2024"},"versionCreatedAt":"2023-11-16 15:50:06","video":"","vorDoi":"10.1007/s10895-023-03580-x","vorDoiUrl":"https://doi.org/10.1007/s10895-023-03580-x","workflowStages":[]},"version":"v1","identity":"rs-3605655","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3605655","identity":"rs-3605655","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}