A new simple method for quantification and locating P and N reserves in microalgal cells based on energy-filtered transmission electron microscopy (EFTEM) elemental maps

Abstract

We established a new simple approach to study phosphorus (P) and nitrogen (N) reserves at subcellular level potentially applicable to various types of cells capable of accumulating P- and/or N-rich inclusions. Here, we report on using this approach for locating and assessing the abundance of the P and N reserves in microalgal and cyanobacterial cells. The approach includes separation of the signal from P- or N-rich structures from noise on the energy-filtered transmission electron microscopy (EFTEM) P- or N-maps. The separation includes (i) relative entropy estimation for each pixel of the map, (ii) binary thresholding of the map, and (iii) segmenting the image to assess the inclusion relative area and localization in the cell section. The separation is based on comparing the a posteriori probability that a pixel of the map contains information about the sample vs. Gaussian a priori probability that the pixel contains noise. The difference is expressed as relative entropy value for the pixel; positive values are characteristic of the pixels containing the payload information about the sample. This is the first known method for quantification and locating at a subcellular level P-rich and N-rich inclusions including tiny (< 180 nm) structures. We demonstrated the applicability of the proposed method both to the cells of eukaryotic green microalgae and cyanobacteria. Using the new method, we elucidated the heterogeneity of the studied cells in accumulation of P and N reserves across different species. The proposed approach will be handy for any cytological and microbiological study requiring a comparative assessment of subcellular distribution of cyanophycin, polyphosphates or other type of P- or N-rich inclusions. An added value is the potential of this approach for automation of the data processing and evaluation enabling an unprecedented increase of the EFTEM analysis throughput.

Phosphorus Feast and Famine in Cyanobacteria: Is Luxury Uptake of the Nutrient Just a Consequence of Acclimation to Its Shortage?

“…The reserve structures were represented by regularly distributed moderately ample α-granules (glycogen), β-granules (lipid droplets), and polyhydroxybutyrate (PHB) granules. On the periphery of the PHB granules close to the nucleoid, and in the interthylakoid space, the electron-dense granules were located, identified by their EDX spectra as P-containing inclusions (PolyP) [ 48 ]. The granules of cyanophycin were small (0.015 ± 0.001 μm 2 on an average) and encountered on less than 60% of the studied cell sections ( Figure 6 ).…”

“…PCC 7118 in transit from P starvation to ample P conditions. We also complemented the traditional transmission electron microscopy (TEM) with analytical TEM, providing reliable information on the subcellular distribution of P-rich inclusions [ 48 ], including polyphosphate formed after the refeeding of P-starving cells [ 58 ]. We leveraged these data to search for specific acclimations to ample P conditions.…”

Wheat Can Access Phosphorus From Algal Biomass as Quickly and Continuously as From Mineral Fertilizer

“…The ability of algae to accumulate nutrients, directly and specifically, make them a valuable vector for recycling nutrients in agricultural systems. Algae can store excess phosphorus (P) and nitrogen (N) inside their cells resulting in high amounts of P and N that can be stored in vacuoles or the cytosol (Ismagulova et al, 2018). Excess supply of phosphate, or similar P sources, can be stored as polyphosphate granules in storage bodies (Miyachi et al, 1964).…”

Subcellular Chemical Imaging: New Avenues in Cell Biology

“…bromo-deoxyurine (BrdU), which is incorporated into replicating DNA during cell division, can be detected by fluorescence immunohistochemistry but also as bromine ions (e.g., 79 Br, 81 Br) by nanoSIMS in the same cells [63]. Fluorine ( 19 F) labelling of proteins using 19 F-azide probe [64] or nm -200 kV) and the elements of interest can be preserved, simultaneous structural and elemental information can be obtained at the nanoscale (Figure 3) [69]. However, molecular information is not available and absolute quantitation can be difficult.…”