Depth of focus extended microscope configuration for imaging of incorporated groups of molecules, DNA constructs and clusters inside bacterial cells

Fessl, Tomáš; Ben-Yaish, Shai; Vacha, František; Adamec, F.; Zalevsky, Zeev

doi:10.1016/j.optcom.2009.03.044

Cited by 5 publications

(2 citation statements)

References 12 publications

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“…The information on the number of molecules per cell was obtained via visual counting. This approach was made possible by exploiting the element that extends the depth of focus of the microscope objective (EDOF), as described in Fessl et al ( 25 ). When using this element, the setup can also detect molecules that are out of focus using the plane microscope objective.…”

Section: Methodsmentioning

confidence: 99%

“…For measurements of the fluorescence lifetimes, a time-correlated single photon counting avalanche photodiode (Perkin Elmer, SPCM-AQR-16) was attached to the side exit of the Triax monochromator (Jobin Yvon Inc., USA). The samples were excited at 640 nm by a total internal reflection prism ( 25 ). Acquired in-cell FRET efficiencies were evaluated in terms of the most likely donor and acceptor positions and their uncertainties using the same procedure as described for in vitro data.…”

Section: Methodsmentioning

confidence: 99%

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Towards characterization of DNA structure under physiological conditions in vivo at the single-molecule level using single-pair FRET

Fessl

Adamec

Polı́vka

et al. 2012

Nucleic Acids Research

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Fluorescence resonance energy transfer (FRET) under in vivo conditions is a well-established technique for the evaluation of populations of protein bound/unbound nucleic acid (NA) molecules or NA hybridization kinetics. However, in vivo FRET has not been applied to in vivo quantitative conformational analysis of NA thus far. Here we explored parameters critical for characterization of NA structure using single-pair (sp)FRET in the complex cellular environment of a living Escherichia coli cell. Our measurements showed that the fluorophore properties in the cellular environment differed from those acquired under in vitro conditions. The precision for the interprobe distance determination from FRET efficiency values acquired in vivo was found lower (∼31%) compared to that acquired in diluted buffers (13%). Our numerical simulations suggest that despite its low precision, the in-cell FRET measurements can be successfully applied to discriminate among various structural models. The main advantage of the in-cell spFRET setup presented here over other established techniques allowing conformational analysis in vivo is that it allows investigation of NA structure in various cell types and in a native cellular environment, which is not disturbed by either introduced bulk NA or by the use of chemical transfectants.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%