Nanopore-Based Optofluidic Devices for Single Molecule Sensing - Nanofluidics: Nanoscience and Nanotechnology - page 151

Figure 7.8 The primary features that make a confocal microscope superior to a conventional microscope

are the pinhole and the objective lens. Only those rays from the best-focus point on the
specimen pass through the pinhole and hit the detector. The light source is usually a laser.

Figure 7.9 depicts a schematic of the nanopore assembly where the membrane is fixed onto
a glass substrate. The bottom reservoir is sealed with a glass coverslip having a thickness
below 200 µm. In this situation, the coverslip is covered with a thin conductive film of
indium tin oxide (ITO) on one of its surfaces. The ITO layer can then be used as the anode.
A spacing layer of controllable thickness prevents any contact between the membrane and
the coverslip. Figure 7.10 shows a photon burst scan of double-stranded -DNA (48 kbp in
length) labelled with YOYO-1 as the sample. YOYO-1 is an intercalating dye and hence
each DNA molecule contains more than nine thousand fluorophores along its backbone,
resulting in a high signal-to-noise ratio. To generate this dataset, the laser was focused on a
single pore and resulting fluorescence photons were counted using an APD. A DC voltage
was applied across the membrane to drive the molecules through the pores. Accordingly,
each photon burst corresponds to a DNA translocation from the top to the bottom reservoir.