In situ quantification of ribosome number by electron tomography.

Ribosomes, discovered in 1955 by George Palade, were initially described as small cytoplasmic particles preferentially associated with the endoplasmic reticulum (ER). Over the years, extensive research has focused on both the structure and function of ribosomes. However, a fundamental question - how many ribosomes are present within whole cells - has remained largely unaddressed. In this study, we developed a microscopic method to quantify the total number of ribosomes in hTERT-RPE-1 cells and in nematode cells from various tissues of Caenorhabditis elegans hermaphrodites. Using electron tomography of high-pressure frozen, freeze-substituted and resin-embedded samples, we determined that the ribosome number in hTERT-RPE-1 cells is in the same order of magnitude as biochemical measurements obtained via RNA capillary electrophoresis. As expected, control worms exhibited a higher number of ribosomes compared to RNA polymerase I A subunit (RPOA-1)-depleted worms in two out of three analysed tissue types. Our imaging-based approach complements established biochemical methods by enabling direct quantification of ribosome numbers in specific samples. This method offers a powerful tool for advancing our understanding of ribosome localisation and distribution in cells and tissues across diverse model systems.