At the single-molecule level, the destruction and/or blinking of a fluorophore upon high-intensity light exposure results in an absent or inconsistent signal from that molecule, which would experimentally be interpreted as a false negative. The imaging solution makes it possible to expose fluorophores to high-intensity laser light for longer periods of time, which results in a sustained fluorescence signal.
Figure 2B, a photobleaching time course experiment, plots the number of single molecules that can be detected over an 80 second exposure time, both in the presence and absence of imaging solution. As shown in the graph, in the absence of solution, over 90% of the single molecules disappear after only 10 seconds of exposure. On the other hand, in the presence of the imaging solution, 95% of the single molecules are detectable after 80 seconds of exposure.
The results from Figure 2 demonstrate the utility of these fluorescently labeled nucleotides for single-molecule imaging and ultimately for use in single-molecule sequencing.
The HeliScope Single Molecule Sequencer can detect the presence of nonspecific, single-molecule adsorption to the surface. When sequencing single-molecule DNA templates, nucleotides that are incorporated into complementary DNA strands might be confounded with nucleotides that adsorb to the surface within a radius of the template equal to the diffraction limit of light.
Since this type of event could result in an insertion error in the sequence, surfaces and surface rinsing conditions were developed that were refractory to nonspecific adsorption and thereby capable of virtually eliminating seemingly spurious incorporation events.