Fig. 1. Schematic of thermal-plex imaging.
a, The thermal-plex imaging concept is based on stepwise melting of DNA thermal probes from an in situ target. After labeling the target biomolecule with a DNA barcode, the thermal probe set comprising a quencher strand and an imager strand is hybridized to the DNA barcode. At temperatures lower than the melting temperature of the quencher, the fluorescent signal is quenched. When the temperature is heated to the signal temperature, the quencher is melted off and fluorescence signal is emitted and can be imaged, including after cooling the sample to room temperature. Signal is then removed by heating to a temperature substantially higher than the melting temperature of the barcode domain (to which the imager binds). b, Thermal spectrum of an exemplary thermal-plex probe set. Ts is the temperature that gives maximum fluorescent signal. Tmq is the melting temperature of the quencher. Tmb is the melting temperature of the barcode domain. The width is the distance between the half maximum of the signal. c, Imaging setup for thermal-plex. Cells are seeded on a slide with an on-scope temperature control module, which can change the slide temperature rapidly in seconds to dissociate DNA strand into the imaging buffer. d, Temperature profile of the slide during the heating and cooling process. It takes about 5 s to reach the desired signal temperature (57 °C) to melt off the DNA strands. After the temperature is cooled down to around 30 °C, the sample is imaged. e, Example process of multiplexed fluorescent imaging for three targets (Ta, Tb and Tc) with thermal-plex. (1) Single-stranded DNA barcodes (a1, b1 and c1) are attached to different target molecules. (2) Orthogonal DNA thermal probe sets (a, b and c) are hybridized to the DNA barcodes. (3) The target Ta is visualized at room temperature after the heating to signal temperature Tsa to activate the fluorescence signal. Several other targets are visualized sequentially after heating to their assigned signal temperatures (Tsb, Tsc). (4) The images are aligned computationally and reconstructed to overlay all the targets.