Single molecule optical measurements of orientation and rotations of biological macromolecules

Shroder, Deborah Y.; Lippert, Lisa G.; Goldman, Yale E.

doi:10.1088/2050-6120/4/4/042004

Cited by 29 publications

(25 citation statements)

References 150 publications

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“…The resulting pores can be tailored to produce diameters in the range of 350–100 nm (Fig 2H), which is essential for constructing waveguides with cutoff wavelengths in the visible range appropriate for experiments with the most commonly used probes for single molecule fluorescence. In addition, a round cross-section is also important; in non-centro-symmetric waveguides, the transmission is polarization sensitive, which could compromise the attenuation of the ZMWs [44] or cause their fluorescence to be sensitive to the orientation of the macromolecules [45].…”

Section: Resultsmentioning

confidence: 99%

Nanoaperture fabrication via colloidal lithography for single molecule fluorescence analysis

et al. 2019

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In single molecule fluorescence studies, background emission from labeled substrates often restricts their concentrations to non-physiological nanomolar values. One approach to address this challenge is the use of zero-mode waveguides (ZMWs), nanoscale holes in a thin metal film that physically and optically confine the observation volume allowing much higher concentrations of fluorescent substrates. Standard fabrication of ZMWs utilizes slow and costly E-beam nano-lithography. Herein, ZMWs are made using a self-assembled mask of polystyrene microspheres, enabling fabrication of thousands of ZMWs in parallel without sophisticated equipment. Polystyrene 1 μm dia. microbeads self-assemble on a glass slide into a hexagonal array, forming a mask for the deposition of metallic posts in the inter-bead interstices. The width of those interstices (and subsequent posts) is adjusted within 100–300 nm by partially fusing the beads at the polystyrene glass transition temperature. The beads are dissolved in toluene, aluminum or gold cladding is deposited around the posts, and those are dissolved, leaving behind an array ZMWs. Parameter optimization and the performance of the ZMWs are presented. By using colloidal self-assembly, typical laboratories can make use of sub-wavelength ZMW technology avoiding the availability and expense of sophisticated clean-room environments and equipment.

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Section: Resultsmentioning

confidence: 99%

Nanoaperture fabrication via colloidal lithography for single molecule fluorescence analysis

et al. 2019

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“…The electromagnetic radiation from a molecular chromophore is proportional to sine-squared (sin 2 ) of the intersecting angle between emission dipole orientation and direction of observation. Mostly the absorption dipole moment orientation and emission dipole moment orientation of fluorophores can be regarded as parallel [4] , [24] , [25] .…”

Section: Principles Of Fpmmentioning

confidence: 99%

“…In Fig. 1 I, simultaneous polarization excitation and polarization detection could be used to analyze the transition dipole moments of absorption and emission, as well as the energy transfer between them [7] , [25] , [37] , [38] . In the defocused pattern recognition (DPR) optical path, circularly polarized light is used for the illumination of all dipoles ( Fig.…”

Section: Principles Of Fpmmentioning

confidence: 99%

Advances of super-resolution fluorescence polarization microscopy and its applications in life sciences

Chen

Yang

et al. 2020

Computational and Structural Biotechnology Journal

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Fluorescence polarization microscopy (FPM) analyzes both intensity and orientation of fluorescence dipole, and reflects the structural specificity of target molecules. It has become an important tool for studying protein organization, orientational order, and structural changes in cells. However, suffering from optical diffraction limit, conventional FPM has low orientation resolution and observation accuracy, as the polarization information is averaged by multiple fluorescent molecules within a diffraction-limited volume. Recently, novel super-resolution FPMs have been developed to break the diffraction barrier. In this review, we will introduce the recent progress to achieve sub-diffraction determination of dipole orientation. Biological applications, based on polarization analysis of fluorescence dipole, are also summarized, with focus on chromophore-target molecule interaction and molecular organization.

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“…In addition, a round cross-section is also important; in non-centrosymmetric waveguides, the transmission is polarization sensitive, which could compromise the attenuation of the ZMWs [38] or cause their fluorescence to be sensitive to the orientation of the macromolecules. [39]…”

Section: Formation and Annealing Of Polystyrene Bead Maskmentioning

confidence: 99%

Nanoaperture fabrication via colloidal lithography for single molecule fluorescence imaging

Jamiolkowski

Chen

Fiorenza

et al. 2019

Preprint

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15In single molecule fluorescence studies, background emission from labeled substrates often restricts 16 their concentrations to non-physiological nanomolar values. One approach to address this challenge is 17 the use of zero-mode waveguides (ZMWs), nanoscale holes in a thin metal film that physically and 18 optically confine the observation volume allowing much higher concentrations of fluorescent substrates. 19Standard fabrication of ZMWs utilizes slow and costly E-beam nano-lithography. Herein, ZMWs are 20 made using a self-assembled mask of polystyrene microspheres, enabling fabrication of thousands of 21 ZMWs in parallel without sophisticated equipment. Polystyrene 1 μm dia. microbeads self-assemble on 22 a glass slide into a hexagonal array, forming a mask for the deposition of metallic posts in the inter-bead 23interstices. The width of those interstices (and subsequent posts) is adjusted within 100-300 nm by 24 partially fusing the beads at the polystyrene glass transition temperature. The beads are dissolved in 25 toluene, aluminum or gold cladding is deposited around the posts, and those are dissolved, leaving 26 behind an array ZMWs. Parameter optimization and the performance of the ZMWs are presented. By 27 using colloidal self-assembly, typical laboratories can make use of sub-wavelength ZMW technology 28 avoiding the availability and expense of sophisticated clean-room environments and equipment. 29 30

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Single molecule optical measurements of orientation and rotations of biological macromolecules

Cited by 29 publications

References 150 publications

Nanoaperture fabrication via colloidal lithography for single molecule fluorescence analysis

Nanoaperture fabrication via colloidal lithography for single molecule fluorescence analysis

Advances of super-resolution fluorescence polarization microscopy and its applications in life sciences

Nanoaperture fabrication via colloidal lithography for single molecule fluorescence imaging

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