Processor for demixing a fluorescent-light input signal, fluorescence microscope and fluorescence microscopy method

A processor for demixing a fluorescent-light input signal of a fluorescence microscope, the fluorescent-light input signal comprising at least two fluorescence emission responses that overlap in time, each of the at least two fluorescence emission responses being representative of an individual impulse response of a fluorophore to a fluorescence-triggering light pulse of a clocked time series of fluorescence-triggering light pulses, the processor being configured to:

• receive a trigger signal comprising a time series of time markers, the trigger signal being representative of a clocking rate, at which the clocked time series of fluorescence-triggering light pulses is generated;
• and
• separate at least one fluorescence emission response from the fluorescent-light input signal by:
• computing at last one decay rest, wherein the at least one decay rest comprises a number of portions of decay tails, and
• subtracting the decay rest from the fluorescent-light input signal, each of the decay tails corresponding to an impulse response generated by a different clocked fluorescence-triggering light pulse.

A non-transitory computer-readable medium having processor-executable instructions stored thereon, wherein the processor-executable instructions, when executed by a processor, facilitates performance of a method for demixing a fluorescent-light input signal of a fluorescence microscope, the fluorescent-light input signal comprising at least two fluorescence emission responses that overlap in time, each of the at least two fluorescence emission responses being representative of an individual impulse response of a fluorophore to a fluorescence-triggering light pulse of a clocked time series of fluorescence-triggering light pulses, the method comprising:

• receiving a trigger signal comprising a time series of time markers, the trigger signal being representative of a clocking rate, at which the clocked time series of fluorescence-triggering light pulses is generated;
• and
• separating at least one fluorescence emission response from the fluorescent-light input signal by at least one of:
• computing at last one decay rest, and subtracting the at least one decay rest from the fluorescent-light input signal, the decay rest being representative of at least one portion of a decay tail of a previous fluorescence emission response;
• linearly unmixing of the fluorescent-light input signal;
• or
• phasor-based unmixing of the fluorescent-light input signal.