Nanometer-accuracy distance measurements between fluorophores at the single-molecule level

Niekamp, Stefan; Sung, Jongmin; Huynh, Walter; Bhabha, Gira; Vale, Ronald D.; Stuurman, Nico

doi:10.1073/pnas.1815826116

Cited by 37 publications

(75 citation statements)

References 49 publications

(118 reference statements)

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“…Flow-cell preparation. Flow-cells were assembled as previously described 27 . Briefly, we made custom three-cell flow chambers using laser-cut double-sided adhesive sheets Assembly and analysis of DNA Fluorocubes.…”

Section: Methodsmentioning

confidence: 99%

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A 6-nm ultra-photostable DNA Fluorocube for fluorescence imaging

Niekamp

Stuurman

Vale

2019

Preprint

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Photobleaching limits extended imaging of fluorescent biological samples. Here, we developed DNA origami-based "Fluorocubes" that are similar in size to the green fluorescent protein (GFP), have single-point attachment to proteins, have a 50-fold higher photobleaching lifetime and emit 40-fold more photons than single organic dyes. We demonstrate that DNA Fluorocubes provide outstanding tools for single-molecule imaging, allowing the tracking of single motor proteins for >800 steps with nanometerprecision.

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Section: Methodsmentioning

confidence: 99%

“…Microscopy' plug-in as previously described 27 . Parameters for analysis are shown in Supplementary Table 10 .…”

Section: "Localizationmentioning

confidence: 99%

A 6-nm ultra-photostable DNA Fluorocube for fluorescence imaging

Niekamp

Stuurman

Vale

2019

Preprint

Self Cite

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“…With Feedback SMLM, such postacquisition processing steps are not required, and yet, structures such as individual F-actin filaments and molecular separation distances <20 nm could be resolved. Feedback SMLM can be further combined with advanced analysis for distance measurements without the need to filter out poorly localized molecules (12,32).…”

Section: Discussionmentioning

confidence: 99%

“…In the second type of experiment, the same molecule is imaged many times to improve the imaging resolution (10,11). These approaches are designed for recombinant samples (11,12) and require extensive post-acquisition processing (12,13). These methods can become inaccurate when distances are similar to the localization precision (<30 nm) (12) and often rely on averaging data over multiple structures and samples (11,13).…”

Section: Introductionmentioning

confidence: 99%

Ultraprecise single-molecule localization microscopy enables in situ distance measurements in intact cells

et al. 2020

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Single-molecule localization microscopy (SMLM) has the potential to quantify the diversity in spatial arrangements of molecules in intact cells. However, this requires that the single-molecule emitters are localized with ultrahigh precision irrespective of the sample format and the length of the data acquisition. We advance SMLM to enable direct distance measurements between molecules in intact cells on the scale between 1 and 20 nm. Our actively stabilized microscope combines three-dimensional real-time drift corrections and achieves a stabilization of <1 nm and localization precision of ~1 nm. To demonstrate the biological applicability of the new microscope, we show a 4-to 7-nm difference in spatial separations between signaling T cell receptors and phosphatases (CD45) in active and resting T cells. In summary, by overcoming the major bottlenecks in SMLM imaging, it is possible to generate molecular images with nanometer accuracy and conduct distance measurements on the biological relevant length scales. Ultraprecise single-molecule localization microscopy enables in situ distance measurements in intact cells. Sci. Adv. 6, eaay8271 (2020).

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“…We corrected for chromatic aberrations, which can introduce errors into CSOP measurements ( Fig. S1) (31), by implementing a calibration procedure before imaging on a new microscope (Fig. S2, Materials and Methods).…”

Section: Validation Of Molecular Height Measurements With Csopmentioning

confidence: 99%

Molecular height measurement by cell surface optical profilometry (CSOP)

Son

Takatori

Belardi

et al. 2020

Preprint

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The physical dimensions of proteins and glycans on cell surfaces can critically affect cell function, for example by preventing close contact between cells and limiting receptor accessibility. However, high-resolution measurements of molecular heights on native cell membranes have been difficult to obtain. Here we present a simple and rapid method that achieves nanometer height resolution by localizing fluorophores at the tip and base of cell surface molecules and determining their separation by radially averaging across many molecules. We use this method, which we call cell surface optical profilometry (CSOP), to quantify height of key multi-domain proteins on a model macrophage and cancer cell, as well as to capture average protein and glycan heights on native cell membranes. We show that average height of a protein is significantly smaller than its contour length due to thermally driven bending and rotation on the membrane and that height strongly depends on local surface and solution conditions. We find that average height increases with cell surface molecular crowding, while it decreases with solution crowding by solutes, both of which we confirm with molecular dynamics simulations. We also use experiments and simulations to determine the height of an epitope based on the location of an antibody, which allows CSOP to profile various proteins and glycans on a native cell surface using antibodies and lectins. This versatile method for profiling cell surfaces has the potential to advance understanding of the molecular landscape of cells and its role in cell function.

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Nanometer-accuracy distance measurements between fluorophores at the single-molecule level

Cited by 37 publications

References 49 publications

A 6-nm ultra-photostable DNA Fluorocube for fluorescence imaging

A 6-nm ultra-photostable DNA Fluorocube for fluorescence imaging

Ultraprecise single-molecule localization microscopy enables in situ distance measurements in intact cells

Molecular height measurement by cell surface optical profilometry (CSOP)

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