Fig. 1: Multi-round smHCR-FISH for archaeal gene expression.

a Schematic of multi-round smHCR-FISH for labeling and imaging mRNA in archaeal cells across various growth stages. In each round, mRNAs from three genes are hybridized with primary probes, whose signals are read through the polymerization of two amplifier probes. After imaging, the targeted probes are cleared by DNase I. b Methylotrophic methanogenesis pathway in Methanococcoides orientis (LMO-1), with non-conventional pathway components in gray. Fmd, formylmethanofuran dehydrogenase; Ftr, formylmethanofuran-H₄MPT formyltransferase; Mch, methenyl-H₄MPT cyclohydrolase; Mtd, F₄₂₀-dependent methylene H₄MPT dehydrogenase; Mer, F₄₂₀-dependent methylene-H₄MPT reductase; Mtr, Na⁺-translocating methyl-H₄MPT-coenzyme-M-methyltransferase; Mcr, methyl-coenzyme M reductase; Fpo, F₄₂₀H₂ dehydrogenase; Hdr, heterodisulfide reductase; MmcA, multi-haem C-type cytochrome; Rnf, Na⁺-translocating ferredoxin-NAD oxidoreductase; F₄₂₀H₂, reduced coenzyme F₄₂₀; H₄MPT, tetrahydromethanopterin; CoB, coenzyme B; CoM, coenzyme M; CoM-S-S-CoB, coenzyme B-coenzyme M heterodisulfide; Fd, ferredoxin, a two-electron carrier; red, reduced; ox, oxidized. c Five-round smHCR-FISH applied to LMO-1 for labeling thirteen protein-coding genes and 16S rRNA. Top: for each round, raw fluorescence data (upper) identify mRNA spots for each gene (lower). Bottom: spots from different genes are overlaid for composite imaging. Each pseudocolor denotes a gene. Scale bars, 2 μm. d Intensity histogram of mcrA smHCR-FISH spots (4438 spots). The typical intensity corresponding to a single mRNA molecule is determined by fitting the histogram to a sum of Gaussians. Inset, smHCR-FISH image of a representative cell. Scale bar, 1 μm. A.u., arbitrary unit. e Average copy number of mcrA mRNA per cell (mean ± s.t.d.) across different growth phases measured by smHCR-FISH and RT-qPCR (n = 3666, 6742, 6375, 7118, 6476, 4329, and 3909 cells per time point from three biological replicates). Error bars, s.t.d. Insets, mcrA signals in different growth phases. Scale bars, 2 μm. f DNA, total RNA, and 16S rRNA signals in log- and stationary-phase cells. The 16S rRNA signal is merged with the identified mRNA spots. Each pseudocolor denotes a gene. Scale bars, 2 μm. c, d, f Experiments were repeated twice with similar results. Source data are provided as a Source Data file.