Microcantilever based distance control between a probe and a surface

Molenaar, Robert; Prangsma, Jord C.; Werf, Kees O. van der; Bennink, Martin L.; Blum, Christian; Subramaniam, Vinod

doi:10.1063/1.4922885

Cited by 4 publications

(7 citation statements)

References 32 publications

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“…We obtain a displacement range from in-contact (d = 0 μm) up to d = 2 μm and an axial positioning accuracy of better than Δd = 3 nm. 40 We validate our experimental approach using the wellcharacterized synthetic fluorophore rhodamine 101, a dye without dark fraction that is often used as a standard reference dye in fluorescence quantum yield measurements. 46 Moreover, Figure 3 shows that it emits in a similar wavelength range as the fluorescent proteins studied here.…”

Section: ■ Resultsmentioning

confidence: 93%

“…The deflection from an in-contact AFM cantilever is used as a feedback signal to control the distance d between the mirror and the sample. We obtain a displacement range from in-contact ( d = 0 μm) up to d = 2 μm and an axial positioning accuracy of better than Δd = 3 nm …”

Section: Resultsmentioning

confidence: 96%

“…To control the distance d between the emitters and the metallic mirror, we used a recently developed method 40 whereby a large (diameter = 100 μm) gold-coated polystyrene sphere serves as a movable mirror. To this end, the sphere is rigidly attached to the stiff base of an AFM microcantilever chip.…”

Section: ■ Resultsmentioning

confidence: 99%

“…The gold-coated sphere was glued to the base of an AFM cantilever. To control the distance between the gold-coated sphere and the fluorophores, and hence the LDOS the fluorophores experience, we used an approach we recently developed; see ref and the schematic in Figure a. In short: We used the deflection from the in contact microcantilever to control the distance between the metallic mirror consisting of a 100 μm gold-coated sphere (see Figure b) and the sample surface.…”

Section: Experimental Sectionmentioning

confidence: 99%

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Quantitative Determination of Dark Chromophore Population Explains the Apparent Low Quantum Yield of Red Fluorescent Proteins

et al. 2020

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The fluorescence quantum yield of four representative red fluorescent proteins mCherry, mKate2, mRuby2, and the recently introduced mScarlet was investigated. The excited state lifetimes were measured as a function of the distance to a gold mirror in order to control the local density of optical states (LDOS). By analyzing the total emission rates as a function of the LDOS, we obtain separately the emission rate and the nonradiative rate of the bright states. We thus obtain for the first time the bright state quantum yield of the proteins without interference from dark, nonemitting states. The bright state quantum yields are considerably higher than previously reported quantum yields that average over both bright and dark states. We determine that mCherry, mKate2, and mRuby2 have a considerable fraction of dark chromophores up to 45%, which explains both the low measured quantum yields of red emitting proteins reported in the literature and the difficulties in developing high quantum yield variants of such proteins. For the recently developed bright mScarlet, we find a much smaller dark fraction of 14%, accompanied by a very high quantum yield of the bright state of 81%. The presence of a considerable fraction of dark chromophores has implications for numerous applications of fluorescent proteins, ranging from quantitative fluorescence microscopy to FRET studies to monitoring protein expression levels. We recommend that future optimization of red fluorescent proteins should pay more attention to minimizing the fraction of dark proteins.

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Section: ■ Resultsmentioning

confidence: 93%

Section: Resultsmentioning

confidence: 96%

Section: ■ Resultsmentioning

confidence: 99%

Section: Experimental Sectionmentioning

confidence: 99%

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Quantitative Determination of Dark Chromophore Population Explains the Apparent Low Quantum Yield of Red Fluorescent Proteins

et al. 2020

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“…Mechanical vibrations, uneven thermal expansion or any mechanical drift easily change the distances in the order of some hundred nanometers. We recently developed a method based on AFM technology to position a probe above a surface with nanometer accuracy [18]. We use the deflection from an in-contact AFM cantilever as feedback signal to achieve realtime control over the distance d between probe and sample [schematic see Fig.…”

Section: Technical Realizationmentioning

confidence: 99%

Photonic emitter manipulation to sample nanoscale topography

et al. 2019

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We demonstrate that photonic emitter manipulation can be used to image the nanoscale topography of a fluorescently labeled layer in confocal imaging. We exploit the fact that a metallic probe manipulates a fluorophore's photonic environment, and thereby its fluorescent lifetime, in a strongly distance-dependent manner. To image surface topography, a metallic probe that is not in contact with the surface is rasterscanned over a fluorescently labeled sample. The axial position of the probe is kept constant. At each lateral probe position, the fluorescence decay is recorded and analyzed to obtain probe -sample distances and hence, the topography of the sample. We present images resolving a microfabricated step of 14 nm in topography, with the probe positioned at different axial positions.

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Quantification of Dark Protein Populations in Fluorescent Proteins by Two-Color Coincidence Detection and Nanophotonic Manipulation

et al. 2022

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Genetically encoded visible fluorescent proteins (VFPs) are a key tool used to visualize cellular processes. However, compared to synthetic fluorophores, VFPs are photophysically complex. This photophysical complexity includes the presence of non-emitting, dark proteins within the ensemble of VFPs. Quantitative fluorescence microcopy approaches that rely on VFPs to obtain molecular insights are hampered by the presence of these dark proteins. To account for the presence of dark proteins, it is necessary to know the fraction of dark proteins (f dark) in the ensemble. To date, f dark has rarely been quantified, and different methods to determine f dark have not been compared. Here, we use and compare two different methods to determine the f dark of four commonly used VFPs: EGFP, SYFP2, mStrawberry, and mRFP1. In the first, direct method, we make use of VFP tandems and single-molecule two-color coincidence detection (TCCD). The second method relies on comparing the bright state fluorescence quantum yield obtained by photonic manipulation to the ensemble-averaged fluorescence quantum yield of the VFP. Our results show that, although very different in nature, both methods are suitable to obtain f dark. Both methods show that all four VFPs contain a considerable fraction of dark proteins. We determine f dark values between 30 and 60% for the different VFPs. The high values for f dark of these commonly used VFPs highlight that f dark has to be accounted for in quantitative microscopy and spectroscopy.

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Microcantilever based distance control between a probe and a surface

Cited by 4 publications

References 32 publications

Quantitative Determination of Dark Chromophore Population Explains the Apparent Low Quantum Yield of Red Fluorescent Proteins

Quantitative Determination of Dark Chromophore Population Explains the Apparent Low Quantum Yield of Red Fluorescent Proteins

Photonic emitter manipulation to sample nanoscale topography

Quantification of Dark Protein Populations in Fluorescent Proteins by Two-Color Coincidence Detection and Nanophotonic Manipulation

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