Dataset Information

Confinement-Free Wide-Field Ratiometric Tracking of Single Fluorescent Molecules.

ABSTRACT: Single-molecule fluorescence has been highly instrumental in elucidating interactions and dynamics of biological molecules in the past two decades. Single-molecule fluorescence experiments usually rely on one of two detection geometries, either confocal point-detection or wide-field area detection, typically in a total internal reflection fluorescence (TIRF) format. However, each of these techniques suffers from fundamental drawbacks that limit their application. In this work, we present a new technique, solution wide-field imaging (SWiFi) of diffusing molecules, as an alternative to the existing methods. SWiFi is a simple extension to existing objective-type TIRF microscopes that allows wide-field observations of fast-diffusing molecules down to single fluorophores without the need of tethering the molecules to the surface. We demonstrate that SWiFi enables high-throughput ratiometric measurements with several thousands of individual data points per minute on double-stranded DNA standard (dsDNA) samples containing Förster resonance energy transfer pairs. We further display the capabilities of SWiFi by reporting on mobility and ratiometric characterization of fluorescent nanodiamonds, DNA Holliday junctions, and protein-DNA interactions. The ability of SWiFi for high-throughput, ratiometric measurements of fast-diffusing species renders it a valuable tool for the single-molecule research community by bridging between confocal and TIRF detection geometries in a simple and efficient way.

SUBMITTER: Gilboa B

PROVIDER: S-EPMC6895709 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Confinement-Free Wide-Field Ratiometric Tracking of Single Fluorescent Molecules.

Gilboa Barak B Jing Bo B Cui Tao J TJ Sow Maabur M Plochowietz Anne A Mazumder Abhishek A Kapanidis Achillefs N AN

Biophysical journal 20191031 11

Single-molecule fluorescence has been highly instrumental in elucidating interactions and dynamics of biological molecules in the past two decades. Single-molecule fluorescence experiments usually rely on one of two detection geometries, either confocal point-detection or wide-field area detection, typically in a total internal reflection fluorescence (TIRF) format. However, each of these techniques suffers from fundamental drawbacks that limit their application. In this work, we present a new t ...[more]

PMID: 31711608

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Project description:When studying microtubules in vitro, label free imaging of single microtubules is necessary when the quantity of purified tubulin is too low for efficient fluorescent labelling or there is concern that labelling will disrupt function. Commonly used techniques for observing unlabelled microtubules, such as video enhanced differential interference contrast, dark-field and more recently laser-based interferometric scattering microscopy, suffer from a number of drawbacks. The contrast of differential interference contrast images depends on the orientation of the microtubules, dark-field is highly sensitive to impurities and optical misalignments. In addition, all of these techniques require costly optical components such as Nomarski prisms, dark-field condensers, lasers and laser scanners. Here we show that single microtubules can be imaged at high speed and with high contrast using interference reflection microscopy without the aforementioned drawbacks. Interference reflection microscopy is simple to implement, requiring only the incorporation of a 50/50 mirror instead of a dichroic in a fluorescence microscope, and with appropriate microscope settings has a similar signal-to-noise ratio to differential interference contrast and fluorescence. We demonstrated the utility of interference reflection microscopy by high-speed imaging and tracking of dynamic microtubules at 100 frames per second. In conclusion, the optical quality of interference reflection microscopy falls within the range of other microscope techniques, being inferior to some and superior to others, depending on the metric used and, with minimal microscope modification, can be used to study the dynamics of unlabelled microtubules.Lay descriptionThe cytoskeleton gives a cell its shape and plays a major role in its movement and division. It's also helps organise the content of cells and is the base for intracellular transport. Important components of the cytoskeleton are microtubules, which are hollow cylindrical beams (25 nm in diameter) that assemble from protein building blocks called tubulin. Deficiencies in microtubules are related to many diseases including cancer and Alzheimer. Given their important role, microtubules are heavily investigated in many laboratories. One way to study microtubules is to isolate them from cells and image them using light microscopy. Over the years a number of imaging techniques have been used. These techniques have a number of drawbacks which are addressed by ongoing efforts which this work is a part of. Here, we present a method based on light interference that produce high quality images of microtubules. The technique is cheap and easy to implement making it accessible to a wide base of researchers.

Dataset Information

Confinement-Free Wide-Field Ratiometric Tracking of Single Fluorescent Molecules.

Publications

Confinement-Free Wide-Field Ratiometric Tracking of Single Fluorescent Molecules.

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