In this report, we adapt and expand in situ hybridization chain reaction (HCR) combined with split-initiator probes to visualize transcripts in the human-pathogenic fungus Candida albicans at high resolution. Investigating this process requires microbial cell-optimized procedures to image and measure mRNAs at single-molecule resolution. Widespread phenomena such as stochastic variation in transcript levels among cells-even within a clonal population-seem to play important roles in the biology of many microorganisms. Yet the accurate quantification of transcripts at single-cell resolution remains challenging for the much smaller microbial cells. IMPORTANCE Tools to visualize and quantify transcripts at single-cell resolution have enabled the dissection of spatiotemporal patterns of gene expression in animal cells and tissues. albicans genes in infected organs at single-cell resolution. The technical development that we introduce, therefore, paves the way to study the patterns of expression of pathogenesis-associated C. The fungus is a major pathogen in humans where it can colonize and invade mucosal surfaces and most internal organs. Finally, we combine HCR with immunostaining to image specific mRNAs and proteins simultaneously within a single C. Furthermore, we establish that HCR is amenable to multiplexing by visualizing different transcripts in the same cell.
mRNA imaging also revealed the subcellular localization of specific transcripts. We show that HCR allows the absolute quantification of transcripts within a cell by microscopy as well as their relative quantification by flow cytometry. Here, we report that in situ hybridization chain reaction (HCR), a method that employs split-initiator probes to trigger signal amplification upon mRNA-probe hybridization, is ideally suited for the imaging and quantification of low-abundance transcripts at single-cell resolution in the fungus Candida albicans. Traditional fluorescence in situ hybridization techniques have been of limited use in fungi due to poor specificity and high background signal. A major disadvantage of this approach is that the transcripts’ spatial distribution and stochastic variation among individual cells within a clonal population is lost. The study of gene expression in fungi has typically relied on measuring transcripts in populations of cells.
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