Fluorescent in-situ hybridization imaging using multiplexed fluorescent switching

A method of fluorescent in-situ hybridization imaging, comprising:

• exposing the sample to a first plurality of first readout probes and a second plurality of second readout probes,
• each first readout probe of the first plurality of first readout probes having a fluorophore to emit light at a first wavelength range and a first targeting portion that binds to a first nucleotide sequence in the sample,
• each second readout probe of the second plurality of second readout probes having a fluorophore to emit light at the first wavelength range and a second targeting portion that binds to a second nucleotide sequence in the sample;
• obtaining a first image of the sample with first readout probes and the second readout probes bound to the first nucleotide sequence and the second nucleotide sequence, respectively, so as to emit light at the first wavelength range;
• treating the sample so as to modify the second readout probes;
• and
• obtaining a second image of the sample with the first readout probes bound to the first nucleotide sequence so as to emit light at the first wavelength range and the second nucleotide sequence substantially not emitting light at the first wavelength range.

A method of fluorescent in-situ hybridization imaging, comprising:

• exposing the sample to a plurality of encoding probes,
• each encoding probe of the plurality of encoding probes having an encoding portion that targets an analyte in the sample and a hybridization portion;
• exposing the sample to a first plurality of first readout probes and a second plurality of second readout probes,
• each first readout probe of the first plurality of first readout probes having a fluorophore to emit light at a first wavelength range and a first targeting portion that binds to a first hybridization sequence in a first encoding probe of the plurality of encoding probes,
• each second readout probe of the second plurality of second readout probes having a fluorophore to emit light at the first wavelength range and a second targeting portion that binds to a second hybridization sequence in a second encoding probe of the plurality of encoding probes;
• obtaining a first image of the sample with the first encoding probes and the second encoding probes having the first readout probes and the second readout probes, respectively, bound thereto so as to emit light at the first wavelength range;
• treating the sample so as to modify the second readout probes on the second plurality of second encoding probes;
• and
• obtaining a second image of the sample with the first encoding probes having the first readout probes bound thereto so as to emit light at the first wavelength range and the second encoding probes substantially not emitting light in at the first wavelength range.

A method of fluorescent in-situ hybridization imaging, comprising:

• exposing the sample to a first plurality of first readout probes and a second plurality of second readout probes,
• each first readout probe of the first plurality of first readout probes having a fluorophore to emit light at a first wavelength range and a first targeting portion that binds to a first analyte in the sample,
• each second readout probe of the second plurality of second readout probes having a fluorophore to emit light at the first wavelength range and a second targeting portion that binds to a second analyte in the sample;
• obtaining a first image of the sample with first readout probes and the second readout probes bound to the first analyte and the second analyte, respectively, so as to emit light at the first wavelength range;
• treating the sample so as to modify the second readout probes;
• and
• obtaining a second image of the sample with the first readout probes bound to the first analyte so as to emit light at the first wavelength range and the second analyte substantially not emitting light at the first wavelength range.