Concentration evaluation of chromatin in unstained resin-embedded sections by means of low-dose ratio-contrast imaging in STEM

Abstract

Quantitative STEM with the imaging mode of ratio-contrast was investigated in order to evaluate the local concentration of DNA in situ for different kinds of DNA plasms in terms of intracellular packing densities (p.d.). The ability of ratio imaging to suppress thickness variations provided the basis to use unstained sections from cryofixed and freeze-substituted material. The DNA p.d. within the nucleoid of E. coli was determined to be about 100 mg ml-1. Quantitative data concerning the p.d. of DNA in condensed eukaryotic chromatin assuming equal amounts of DNA and protein were evaluated for the first time: approximately 400 mg ml-1 chromatin which corresponds to 200 mg ml-1 DNA. The p.d. of DNA in chromosomes from the dinoflagellate Amphidinium carterae, a eukaryote devoid of histones and with only small relative amounts of histone-like protein, was also found to be of the order of 200 mg ml-1. The highest p.d. of DNA was measured for the head of the bacteriophage T4 with more than 800 mg ml-1, in fair agreement with previous calculations. The results provide further support for a condensation mode of low protein chromatins that involves a liquid-crystalline organization of the DNA filaments.