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Our single molecule experiments were carried out over a course of up to one hour.
2D single molecule experiments were conducted on a Nikon Eclipse Ti microscope equipped with a 100× oil-immersion objective lens (Nikon, N.A. = 1.4), a lumencor light source, two filter wheels (Lambda 10-3, Sutter Instrument, Novato, CA), perfect focusing systems, and EMCCD (iXon3, Andor, UK).
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However, forced separations in single molecule experiments are difficult, and therefore molecular simulation techniques were applied in our study.
Here we demonstrate that single molecule imaging in solution and especially in living tissue substantially profits from using light sheet illumination, because single molecule experiments are extremely sensitive in terms of background fluorescence and photobleaching.
The target RNAs used in the ensemble and the single molecule experiments are different from each other in several respects.
However, the dynein concentration in single molecule experiments is most likely much lower than in sliding experiments, so the lack of aggregates in single molecule experiments still doesn't rule out that there are clusters in the sliding assay.
Single-molecule experiments were performed on a custom-built confocal microscope.
Single-molecule experiments were performed with magnetic tweezers (Strick et al, 1996).
Single-molecule experiments were performed with prism-type total internal reflection microscopy.
Single-molecule experiments were performed at room temperature using an inverted wide-field microscope equipped with a 63× NA 1.4 objective lens employing three fluorescence channels (24, 25).
As we indicate in the text, the single-molecule experiments were performed with SSB labeled with AF488, while the equilibrium titrations were performed with SSB labeled with 5,6-carboxyflourescein.
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