Controlling fluorescent signal composition in optically-based nanopore sequencing

A method of determining a nucleotide sequences of polynucleotides, the method comprising the steps of:

• (a) translocating single stranded polynucleotides through nanopores at a translocation speed, wherein nucleotides of the single stranded polynucleotides are labeled with fluorescent labels such that, when exposed to fluorescence excitation light, fluorescent labels of adjacent nucleotides are in a mutually quenched state in free solution and wherein the nanopores force the fluorescent labels within the nanopore into a constrained state wherein substantially no detectable signal is generated;
• (b) exciting the fluorescent labels of nucleotides upon exiting the nanopores and prior to being quenched by the label of an adjacent nucleotide;
• (c) measuring fluorescent signals generated by the fluorescent labels exiting the nanopores and determining from the measured fluorescent signals whether a single fluorescent label contributes to the measured fluorescent signal or a plurality of fluorescent labels contributes to the measured fluorescent signal at each nanopore;
• (d) reducing the translocation speed by a predetermined value whenever a measured fluorescent signal at a nanopore is generated by a plurality of fluorescent labels;
• (e) repeating steps (a) through (d) until the translocation speed corresponds to substantially every measured fluorescent signal at each nanopore comprising fluorescence from substantially a single fluorescent label;
• and
• (f) identifying nucleotides of the translocating polynucleotides from measured fluorescent signals.