ExperimentType multiplexed FISH

Multiplexed FISH

Multiplexed FISH methods are high-throughput, sequential imaging experiments that use fluorophore-labelled oligonucleotides (imaging probes) on fixed samples. By repeating many cycles of (1) imaging probe hybridization, (2) signal detection and (3) signal removal, a high number of targets can be imaged.

These experiments are often multi-modal, targeting different molecule species simultaneously: libraries of primary oligonucleotide probes (e.g. Oligopaints) can be designed to target many genomic loci at different resolutions, or a large number of RNA transcripts, while oligonucleotide-conjugated antibodies can target proteins. Regardless of the target, all primary probes contain oligonucleotide handles that can be recognized by imaging probes during the sequential imaging routines. Multiplexing can be further improved by using multiple fluorophores and combinatorial barcoding schemes.

These techniques can be applied to study the spatial organization of the genome (e.g. chromatin tracing), its transcriptional output, as well as its spatial proximity to nucleoli, nuclear speckles, nuclear lamina, etc. Multiplexed FISH technologies include: MERFISH, MINA, OligoFISSEQ, OligoSTORM, ORCA, seqFISH+, and others.