Analysis of individual (macro)molecules and proteins using near-field optics

Hulst, Niek F. van; Veerman, J.A.; García‐Parajó, María F.; Kuipers, L.

doi:10.1063/1.481385

Cited by 102 publications

(86 citation statements)

References 87 publications

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“…While the far-field generated by the aperture (i.e., for kR ≫ 1) is still in a good approximation described by a dipolar behavior, as confirmed experimentally [44] and theoretically [45,46], the near-field deviates strongly from this simplified assumption. This has been confirmed in several studies [5,7,[47][48][49][50]). Therefore, another approach must be developed.…”

Section: The Aperture Nsom Tipsupporting

confidence: 71%

Near-field microscopy with a single-photon point-like emitter: Resolution versus the aperture tip?

Drezet¹,

Cuche²,

Huant³

2011

Optics Communications

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We discuss theoretically the concept of spatial resolution in near-field scanning optical microscopy (NSOM) in light of a recent work [Opt. Express 17 (2009) 19969] which reported on the achievement of active tips made of a single ultrasmall fluorescent nanodiamond grafted onto the apex of a substrate tip and on their validation in NSOM imaging. Since fluorescent nanodiamonds tend to decrease steadily in size, we assimilate a nanodiamond-based tip to a point-like single photon source and compare its ultimate resolution with that offered by standard metal-coated aperture NSOM tips. We demonstrate both classically and quantum mechanically that NSOM based on a point-like tip has a resolving power that is only limited by the scan height over the imaged system whereas the aperture-tip resolution depends critically on both the scan height and aperture diameter. This is a consequence of the complex distribution of the electromagnetic field around the aperture that tends to artificially duplicate the imaged objects. We show that the point-like tip does not suffer from this "squint" and that it rapidly approaches its ultimate resolution in the near-field as soon its scan height falls below the distance between the two nano-objects to be resolved.

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Section: The Aperture Nsom Tipsupporting

confidence: 71%

Near-field microscopy with a single-photon point-like emitter: Resolution versus the aperture tip?

Drezet¹,

Cuche²,

Huant³

2011

Optics Communications

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“…The fluorescence emission of the individual proteins was collected by a Zeiss 1.3 N.A.͞ϫ63 oil-immersion objective and, after selection by suitable fluorescence filters ( det Ͼ 590 nm), it was separated in two perpendicular polarization components and focused onto two avalanche photodiodes (SPCM-100, EG & G, Quebec, Canada). Details about the experimental setup can be found elsewhere (19). Integration time for imaging was 5 ms͞pixel.…”

Section: Methodsmentioning

confidence: 99%

“…In this technique, an aperture probe of dimensions smaller that the excitation wavelength is used to illuminate the sample (19). Light from a Ar ϩ -Kr ϩ ion laser was coupled to the back side of the NSOM probe (single-mode fiber, ϭ 630 nm, Cutz, Frankfurt, Germany).…”

Section: Methodsmentioning

confidence: 99%

The nature of fluorescence emission in the red fluorescent protein DsRed, revealed by single-molecule detection

García-Parajo

Koopman

Dijk

et al. 2001

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

106

101

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Recent studies on the newly cloned red fluorescence protein DsRed from the Discosoma genus have shown its tremendous advantages: bright red fluorescence and high resistance against photobleaching. However, it has also become clear that the protein forms closely packed tetramers, and there is indication for incomplete protein maturation with unknown proportion of immature green species. We have applied single-molecule methodology to elucidate the nature of the fluorescence emission in the DsRed. Real-time fluorescence trajectories have been acquired with polarization sensitive detection. Our results indicate that energy transfer between identical monomers occurs efficiently with red emission arising equally likely from any of the chromophoric units. Photodissociation of one of the chromophores weakly quenches the emission of adjacent ones. Dual color excitation (at 488 and 568 nm) single-molecule microscopy has been performed to reveal the number and distribution of red vs. green species within each tetramer. We find that 86% of the DsRed contain at least one green species with a red-to-green ratio of 1.2-1.5. On the basis of our findings, oligomer suppression would not only be advantageous for protein fusion but will also increase the fluorescence emission of individual monomers.T he cloning of the red fluorescent protein (drFP583, commercially available as DsRed) from the Indo pacific reef coral Discosoma sp (1) has triggered intense biological interest as a potential expression marker and fusion partner that would be complementary to the Aequorea victoria green fluorescent protein (avGFP). The main advantage of these naturally fluorescent proteins is that they provide strong visible fluorescence and can be genetically fused to other proteins. Since the cloning of the avGFP (2, 3), different mutants have been produced to extend the palette of available colors. However, no GFP mutant has been produced with emission maxima longer than 529 nm (3). The newly cloned DsRed has bright red fluorescence with emission maxima at 583 nm, one of the longest wavelength emissions reported so far in a wild-type species [Fradkov et al. (4) have reported a wild type highly homologous to the drFP583 with 593-nm emission and a hybrid with 616-nm emission]. Potentially, DsRed is an ideal partner of GFP in fluorescence resonance energy transfer (FRET) experiments, particularly in cell biological applications in which cellular autofluorescence poses a problem (5).In the last few months, extensive research has been done in the biochemical and photophysical investigation of DsRed. The absorption spectrum of the mature protein exhibits a strong band with a peak at 558 nm and two minor shoulders at 526 and 490 nm (6). An extinction coefficient of 75,000 mol Ϫ1 ͞cm Ϫ1 and a fluorescence quantum yield of 0.7 at 558-nm excitation wavelength have been reported (7), much higher than initially published by Matz et al. (1). The protein proves to be stable under harsh pH conditions and is extremely resistant to photobleaching (7, 8). However, t...

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“…SNOM looks particularly suitable for this purpose due to the strong localization of the field emitted by the probe; moreover, due to the presence of transverse and longitudinal field components close to the probe apex, it has been used for the determination of the three-dimensional (3D) orientation of single fluorophores by controlling the polarization of the excitation light [29]. Aperture SNOM probes have been employed for fluorescence studies on biological molecules and polymers [24,[28][29][30][31].…”

Section: Introductionmentioning

confidence: 99%

Interaction of an Asymmetric Scanning Near Field Optical Microscopy Probe With Fluorescent Molecules

Lotito

Sennhauser²,

Hafner³

et al. 2011

PIER

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Abstract-We present a numerical analysis of the interaction between novel scanning near field optical microscopy probes based on an asymmetric structure and a single fluorescent molecule. Our finite element analysis shows how such near field probes can be effectively used for high resolution detection of single molecules, in particular those with a longitudinal dipole moment. At the same time, fluorescent molecules can be exploited as point-like probes of the single vectorial components of the near field distribution at the probe apex, providing a powerful tool for near field probe characterization.

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Analysis of individual (macro)molecules and proteins using near-field optics

Cited by 102 publications

References 87 publications

Near-field microscopy with a single-photon point-like emitter: Resolution versus the aperture tip?

Near-field microscopy with a single-photon point-like emitter: Resolution versus the aperture tip?

The nature of fluorescence emission in the red fluorescent protein DsRed, revealed by single-molecule detection

Interaction of an Asymmetric Scanning Near Field Optical Microscopy Probe With Fluorescent Molecules

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