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Researchers Use TIRF Microscopy to Rapidly Record Fluorescence Images from Nanopore Membrane

A team of researchers in the USA has, for the first time, been able to demonstrate the feasibility of high-speed optical identification of individual converted DNA bases as they translocate through solid state nanopores with high temporal resolution (1000 frames per second)1.

The team, which is led by Amit Meller, Associate Professor of Biomedical Engineering and Physics, Boston University, based their work on a custom total internal reflection fluorescence (TIRF) microscopy set-up, incorporating an ultra-sensitive Andor iXon 860 EMCCD camera to rapidly record fluorescence images from the nanopore membrane2.

To harness this powerful single-molecule detection modality for high-throughput DNA sequencing, each of the four nucleotides (A, C, G and T) in the target DNA has to be converted into a predefined sequence of oligonucleotides, using a technique called Circular DNA Conversion. Each pre-defined sequence is then hybridized to a self-quenching molecular beacon.

In their experimental setup, the Meller team recorded DNA translocation through a 4nm pore in a SiN membrane immersed between two aqueous solutions. With the camera in place, the researchers were able to detect florescence bursts in two different colours from individual beacons as they were stripped off the translocating strand, which in turn provides sequence information.

Prof. Meller believes the applicability of this technique has major implications for future approaches to DNA sequencing, “At between 50-250 bases per second, our DNA readout speed is already faster than other single molecule methods, but we believe we can push this up to greater than 500 bases per second by adapting the technique for 4-colour analysis and optimizing the reagents. After that, since our readout process does not involve an enzyme, the speed per nanopore will be determined by the limits of detection offered by state of the art CCD or CMOS technologies, and is readily controlled by the voltage we apply on the SiN membrane. As soon as progress is made in raising imaging speeds, we can immediately utilize in our system, further increasing the readout rates.”

Prof Meller adds that, “The use of highly sensitive and ultra-fast EMCCD is central to our sequencing method, as we rely on fast multi-colour optical readout, from many nanopores simultaneously. The iXon DU-860 currently offers an optimal format for our sequencing technology. Its 24µm size pixels allows an efficient light collection, combined with the >500 fps readout speed for full frame, or higher rates for sub-images. In addition, its low readout noise and very high EM gain are both features that we use extensively in our system. Finally, the back illumination version provides extremely high quantum yield, which is extremely beneficial for high-speed single molecule detection.”

The wide-field optical detection approach used by Meller and his team has an inherent advantage in that numerous pores can be probed simultaneously. This makes it fully scalable and the ideal basis for the next generation of commercial sequencing systems.

1McNally, B., A. Singer, Z. Yu, Y. Sun, Z. Weng, and A. Meller. 2010. Optical Recognition of Converted DNA Nucleotides for Single-Molecule DNA Sequencing Using Nanopore Arrays. Nano Letters 10, 2237-2244.

2Soni, V. G., A. Singer, Z. Yu, Y. Sun, B. McNally, and A. Meller. 2010. Synchronous optical and electrical detection of bio-molecules traversing through solid-state nanopores. Rev. Sci. Instru. 81, 014301-307.

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