Dual-Color Single-Molecule Imaging with MT-TIRF

Experiments were performed in two MT-TIRF setups similar to one described before (Madariaga-Marcos et al., 2018 (link)). In brief, 488 nm laser source (Vortran Stradus) was focused on the back focal plane of a high numerical aperture objective (Olympus UAPON TIRF 100×). We used two separate detectors to visualise the emission of the fluorophores in the sample and the magnetic beads; an EM-CCD temperature-controlled camera (Andor Ixon Ultra 897/Andor Ixon Ultra 888) and a CCD or CMOS camera (Pulnix 6710 CL/Mikrotron MC1362) for bright-field video microscopy. Lateral Magnetic Tweezers consisted of a pair of permanent magnets (Q-05-05-02-G, Supermagnete) connected to a linear motor (Piezomotor). The MT setup was controlled by a custom-written code in Umbarger et al. (2011) (link). Fluorescence camera was controlled by Andor Solis software.

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Madariaga-Marcos J., Pastrana C.L., Fisher G.L., Dillingham M.S, & Moreno-Herrero F. (2019). ParB dynamics and the critical role of the CTD in DNA condensation unveiled by combined force-fluorescence measurements. eLife, 8, e43812.

Publication 2019

UAPON TIRF 100×). Ultra 897

Cmos Fluorescence Video microscopy

Corresponding Organization : Consejo Superior de Investigaciones Científicas

Other organizations : University of Bristol

Top 5 similar protocols

Variable analysis

independent variables

Lateral Magnetic Tweezers consisting of a pair of permanent magnets connected to a linear motor

dependent variables

Emission of the fluorophores in the sample
Magnetic beads

control variables

488 nm laser source focused on the back focal plane of a high numerical aperture objective
EM-CCD temperature-controlled camera and a CCD or CMOS camera for bright-field video microscopy
MT setup controlled by a custom-written code
Fluorescence camera controlled by Andor Solis software

controls

No positive or negative controls were explicitly mentioned in the provided information.

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