The ability to characterise elements, isotopes, and metabolites at the cellular level provides powerful insight into cellular function and responses to biotic and abiotic factors. Here I discuss the use of cryo-scanning electron microscopy (cryoSEM) and energy dispersive spectroscopy (EDS) to map the distribution of elements at the cellular level in various frozen-hydrated plant tissues. From these map data we have been able to quantify cellular element concentrations and to gain valuable insight into plant function and adaptation.
Sample preparation is the most important factor in any analytical method. I will outline the pros and cons of options for cryo-preserving elements of interest (e.g. high pressure freezing, plunge freezing), and highlight the importance of key factors (e.g. flat samples, pulse pile up correction) when aiming to achieve fully quantified concentrations from biological EDS data in the cryoSEM.
Our existing cryoSEM-EDS analytical system includes a cryomicrotome and sample preparation system (Leica) and a field emission SEM (Zeiss), fitted with a cryostage and an 80 mm EDS SDD detector (Oxford Instruments). For analysis of frozen-hydrated samples in the cryoSEM, we cryoplane plunge-frozen samples to generate a flat surface, coat with up to 20 nm Cr, and transfer to the cryoSEM without sublimation. We perform all of our analyses at 15 kV and a 2 nA beam current. We use AZtec software (Oxford Instruments) to conduct all of our analyses and use their in-built standards to perform our quantitation (Marshall, 2017).
Examples presented will include salinity tolerance (Kotula et al. 2019), low-phosphorus adaptation and calcium toxicity (Guilherme Pereira et al. 2018; Hayes et al. 2018; 2019), and iron metabolism.