Visualising individual green fluorescent proteins with a near field optical microscope

García-Parajo, María F.; Veerman, J.A.; Segers-Nolten, Gezina M.J.; Grooth, B.G. de; Greve, Jan; Hulst, Niek F. van

doi:10.1002/(sici)1097-0320(19990701)36:3<239::aid-cyto14>3.0.co;2-y

Cited by 32 publications

(19 citation statements)

References 30 publications

(51 reference statements)

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“…Investigation of the photophysical properties has been carried out in ensemble measurements, at room and low temperatures (7,(11)(12)(13)(14), and at the single-molecular level (15)(16)(17)(18)(19). When molecules are observed individually, the fluorescence emission of GFP shows intensity fluctuations, on-off blinking, and fluorescence switching, a behavior that is hidden in ensemble experiments.…”

mentioning

confidence: 58%

“…Fluorescence correlation spectroscopy has recently shown that typical triplet-state lifetimes of the GFP are 5-25 s (34, 35). The length of the dark periods observed by us and others (15)(16)(17)(18)(19) is on the order of seconds and indicates that the nonemissive state is different from the triplet state. Nevertheless, our observations at different excitation conditions prove that blinking is photoinduced, with dynamics that resemble that of the singlet-triplet system.…”

Section: Light Intervals As a Function Of Excitation Intensitymentioning

confidence: 99%

“…Both techniques allow recording of real-time f luorescence trajectories of individual molecules at different excitation conditions over a wide dynamic range with microsecond time resolution (27,28). In addition, NSOM provides higher spatial resolution and correlated topographic information (18,29). Our experiments show conclusively that excitation intensity has a dramatic effect on GFP blinking, with a reduction of the fraction of molecules being in the ''on'' state when the excitation intensity is increased.…”

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confidence: 99%

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Real-time light-driven dynamics of the fluorescence emission in single green fluorescent protein molecules

García-Parajo

Segers-Nolten²,

Veerman

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

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Real-time single-molecule fluorescence detection using confocal and near-field scanning optical microscopy has been applied to elucidate the nature of the ''on-off'' blinking observed in the Ser-65 3 Thr (S65T) mutant of the green fluorescent protein (GFP). Fluorescence time traces as a function of the excitation intensity, with a time resolution of 100 s and observation times up to 65 s, reveal the existence of a nonemissive state responsible for the long dark intervals in the GFP. We find that excitation intensity has a dramatic effect on the blinking. Whereas the time during which the fluorescence is on becomes shorter as the intensity is increased, the off-times are independent of excitation intensity. Statistical analysis of the on-and off-times renders a characteristic off-time of 1.6 ؎ 0.2 s and allows us to calculate a transition yield of Ϸ0.5 ؋ 10 ؊5 from the emissive to the nonemissive state. The saturation excitation intensity at which on-and off-times are equal is Ϸ1.5 kW͞cm 2 . On the basis of the single-molecule data we calculate an absorption cross section of 6.5 ؋ 10 ؊17 cm 2 for the S65T mutant. These results have important implications for the use of the GFP to follow dynamic processes in time at the single-molecular level. Since the cloning and subsequent expression of the green fluorescent protein (GFP) from the jellyfish Aequorea victoria (1, 2) the research interest for this protein has increased dramatically. The protein has been used successfully in a large and ever-growing number of applications, including gene expression and cell dynamics (3, 4). It is the only cloned protein that exhibits strong intrinsic fluorescence without the need of external chromophores. In the native protein [wild-type (wt)-GFP] the chromophore is formed in an autocatalytic, posttranslational cyclization and oxidation of the tripeptide unit at residues 65-67 (2, 5, 6). The wt-GFP absorbs blue light at Ϸ395 nm, with a weak peak at Ϸ475 nm, and emits green light at Ϸ508 nm (7). Substitution of one or more amino acids at or in close proximity to the chromophore results in mutants with different absorption and emission properties, and in some cases, improved emission and photostability (5,8,9). A widely used mutant is the S65T, in which Ser-65 is replaced by Thr (10). The mutant shows only an absorption peak at Ϸ475 nm, has larger absorption cross section than wt-GFP, and shows no photo-isomerization (8, 9).Because of the rapidly increasing number of applications, great attention has been focused on the photophysical properties of the wt-GFP and a number of its mutants. Investigation of the photophysical properties has been carried out in ensemble measurements, at room and low temperatures (7,(11)(12)(13)(14), and at the single-molecular level (15)(16)(17)(18)(19). When molecules are observed individually, the fluorescence emission of GFP shows intensity fluctuations, on-off blinking, and fluorescence switching, a behavior that is hidden in ensemble experiments. These intriguing phenomena also manifest in many other...

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mentioning

confidence: 58%

Section: Light Intervals As a Function Of Excitation Intensitymentioning

confidence: 99%

mentioning

confidence: 99%

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Real-time light-driven dynamics of the fluorescence emission in single green fluorescent protein molecules

García-Parajo

Segers-Nolten²,

Veerman

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

171

134

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“…At the same time, other detecting devices cannot be excluded such as other types of molecular scanning microscopes [22] or electrochemical detectors on nanowires [23] and nanopores [24].…”

Section: Using the 2-d-lc-ms/ms Method We Could Not Identifymentioning

confidence: 99%

AFM fishing nanotechnology is the way to reverse the Avogadro number in proteomics

et al. 2007

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Future development of proteomics may be hindered by limitations in the concentration sensitivity of widespread technological approaches. The concentration sensitivity limit (CSL) of currently used approaches, like 2-DE/LC separation coupled with MS detection, etc., varies from 10 29 to 10 212 M. Therefore, proteomic technologies enable detection of up to 20% of the protein species present in the plasma. New technologies, like atomic force microscopy (AFM molecular detector), enable the counting of single molecules, whereas biospecific fishing can be used to capture these molecules from the biomaterial. At the same time, fishing also has thermodynamic limitations due to the reversibility of the binding. In cases where the fishing becomes irreversible, its combination with an AFM detector enables the registration of single protein molecules, and that opens up a way to lower the CSL down to the reverse Avogadro number.

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“…Betzig and Chichester [135] were among the first pioneers to investigate biological tissues using SNOM. SNOM potential for the revelation of single molecules was demonstrated on the (S65T) mutant of the GFP [136]. GFP is considered by these authors as an individual marker for applications in molecular biology for detailed understanding of its photophysical and photodynamic properties.…”

Section: Potentialities Of Confocal and Near-field Microscopiesmentioning

confidence: 99%

Nanotechnologies in proteomics

Ivanov

Govorun

Быков³

et al. 2006

Proteomics

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Progress in proteomic researches is largely determined by development and implementation of new methods for the revelation and identification of proteins in biological material in a wide concentration range (from 10 23 M to single molecules). The most perspective approaches to address this problem involve (i) nanotechnological physicochemical procedures for the separation of multicomponent protein mixtures; among these of particular interest are biospecific nanotechnological procedures for selection of proteins from multicomponent protein mixtures with their subsequent concentration on solid support; (ii) identification and counting of single molecules by use of molecular detectors. The prototypes of biospecific nanotechnological procedures, based on the capture of ligand biomolecules by biomolecules of immobilized ligate and the concentration of the captured ligands on appropriate surfaces, are well known; these are affinity chromatography, magnetic biobeads technology, different biosensor methods, etc. Here, we review the most promising nanotechnological approaches for selection of proteins and kinetic characterization of their complexes based on these biospecific methods with subsequent MS/MS identification of proteins and protein complexes. Two major groups of methods for the analysis and identification of individual molecules and their complexes by use of molecular detectors will be reviewed: scanning probe microscopy (SPM) (including atomic-force microscopy) and cryomassdetector technology.

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Visualising individual green fluorescent proteins with a near field optical microscope

Cited by 32 publications

References 30 publications

Real-time light-driven dynamics of the fluorescence emission in single green fluorescent protein molecules

Real-time light-driven dynamics of the fluorescence emission in single green fluorescent protein molecules

AFM fishing nanotechnology is the way to reverse the Avogadro number in proteomics

Nanotechnologies in proteomics

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