Fig. 1.

Schematic of the PRISM imaging technique. a Reagents (markers, shown in gray) for detecting subcellular targets include antibodies or peptides that are conjugated with unique oligonucleotide barcodes (docking strands, shown in blue). A barcoded marker is imaged using the complementary fluorophore-conjugated oligonucleotides (imaging probes) that bind to the docking strands on the marker (see (iii) in b, fluorophores shown as red circles). Binding affinity of the imaging probes to the docking strands can be varied by changing the sequence and type of the oligonucleotides, which thereby enables either diffraction-limited (high affinity) or super-resolution microscopy (low affinity). Conjugation of docking strands to markers using site-specific click chemistry enables stoichiometric control of the number of nucleic acids bound to a whole antibody, while SMCC enables conjugation of docking strands to free amine groups on a variety of markers. b The reagent testing and validation phase consists of (i) generation of reference staining patterns of all molecular targets of interest using standard immunofluorescence (IF), (ii) analysis of the specificity and staining quality of markers conjugated with docking strands compared to those in the reference IF, and (iii) co-localization of PRISM-imaged staining patterns using imaging probes (red circles, which correspond to fluorophores conjugated to the probes) with standard IF staining patterns (green circles). c Overview of the main steps in the PRISM imaging workflow. All molecular targets of interest are immunostained at once using docking strand-conjugated markers (e.g., antibodies shown in green, blue, and pink). Nucleic acid imaging probes specific to each marker (e.g., p₁–p₁₀) are applied and imaged sequentially, with each imaging strand washed out after image acquisition at each step. This approach enables imaging a dozen or more distinct molecular targets in the same sample. Images of different markers are drift-corrected and overlaid to generate a pseudo-colored, multiplexed image. For super-resolution PRISM, prior to drift correction, the super-resolved image of each marker is reconstructed from the temporal image stack of binding/unbinding events of the imaging probes to/from the docking strands on the marker