MERFISH Barcode Decoding | mysimulator.uk

Imaging Rounds

Barcode length N (bits) 12

Per-round bit-error rate 6%

Imaging speed 1.5 rounds/s

Decoding Cycle

Imaging round 0 / 12

Molecules / codewords

800 / 8

Min. Hamming distance

d ≥ 4

Decode accuracy

Error-corrected

Misidentified

No-call (dropped)

How it works

MERFISH (multiplexed error-robust FISH) identifies thousands of RNA species in intact tissue without sequencing. Each gene is assigned a binary barcode of length N: an N-round sequence of hybridize → image → strip cycles, where round i's fluorescent readout probe lights up a molecule only if bit i of its barcode is 1.

Barcode:     b = (b1, b2, ..., bN),  bi ∈ {0,1}
Fixed weight: Σ bi = 4  (balances brightness across rounds)
Code distance: d(gene A, gene B) ≥ 4  for every pair
Correctable errors: floor((d-1)/2) = 1 bit per molecule

Because every valid codeword sits at Hamming distance ≥ 4 from every other, a single imaging error (a false drop-out or a spurious blink) still leaves the corrupted observation closer to the true codeword than to any other — so nearest-codeword decoding recovers the correct identity. Two or more errors on the same molecule can push it past the correction radius, producing a misidentification or an ambiguous no-call.

Barcode length N — more rounds mean a larger, more separated codebook (more distinguishable genes) at the cost of more imaging time.
Bit-error rate — models real dropout from incomplete probe binding, photobleaching, or segmentation noise.
Error correction toggle — with it off, only an exact bit-for-bit match is accepted; with it on, the decoder accepts the nearest codeword within 1 bit, exactly as real MERFISH pipelines do.

Real-world relevance: this combinatorial, error-corrected barcoding is what lets imaging-based spatial transcriptomics profile hundreds to thousands of genes per cell at single-molecule resolution, directly in fixed tissue.

Bit = 1 this round (bright) Bit = 0 this round (dim) Correctly decoded gene No-call / dropped

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