Improvements in biological X-ray microanalysis: cryoembedding for specimen preparation and multivariate statistical analysis for data interpretation.
For biological X-ray microanalysis, cryoembedding (CE) combined with cryofixation (CF) and cryodehydration (CD) was already proposed as an alternative method to freeze-dried cryosections in 1984 by Wróblewski and Wroblewski. CD by freeze-drying (FD) is usually recommended because it provides better retention of diffusible elements. CD by freeze-substitution (FS) has the advantage of being simpler, giving more reproducible preservation of ultrastructure and causing fewer problems for resin infiltration. We have increased the retention of diffusible elements by using home-made devices for CS and CE in the new Lowicryl K11M and HM23 resins. These resins allow samples to be kept at a maximum temperature of 213K and 193K respectively. Application of multivariate statistical analysis (MSA) to X-ray data (spectra and maps) allows the study of correlations between the analyzed elements in different nuclear areas and in the cytoplasm. The "factorial" images, obtained with MSA, display the compartments of strong correlation between P and K (nucleic acids) and the compartments of strong correlation between S and K (proteins). We suggest that the future application of MSA methods will provide increased knowledge of the physio-pathological compartmentation of diffusible elements at the subcellular level.