Subcellular localisation and identification of single atoms using quantitative scanning transmission electron microscopy

Abstract

Determining the concentration of elements in subcellular structures poses a significant challenge. By locating an elemental species at high spatial resolution and with subcellular context, and subsequently quantifying it on an absolute scale, new information about cellular function can be revealed. Such measurements have not as yet been realised with existing techniques due to limitations on spatial resolution and inherent difficulties in detecting elements present in low concentrations. In this paper, we use scanning transmission electron microscopy (STEM) to establish a methodology for localising and quantifying high-Z elements in a biological setting by measuring elastic electron scattering. We demonstrate platinum (Pt) deposition within neuronal cell bodies following in vivo administration of the Pt-based chemotherapeutic oxaliplatin to validate this novel methodology. For the first time, individual Pt atoms and nanoscale Pt clusters are shown within subcellular structures. Quantitative measurements of elastic electron scattering are used to determine absolute numbers of Pt atoms in each cluster. Cluster density is calculated on an atoms-per-cubic-nanometre scale, and used to show clusters form with densities below that of metallic Pt. By considering STEM partial scattering cross-sections, we determine that this new approach to subcellular elemental detection may be applicable to elements as light as sodium. LAY DESCRIPTION: Heterogeneous elemental distributions drive fundamental biological processes within cells. While carbon, hydrogen, oxygen and nitrogen comprise by far the majority of living matter, concentrations and locations of more than a dozen other species must also be tightly controlled to ensure normal cell function. Oxaliplatin is a first-line and adjuvant treatment for colorectal cancer. However, pain in the body's extremities (fingers and toes) significantly impairs clinical usage as this serious and persistent side effect impacts on both patient cancer care and quality of life. Annular dark-field (ADF) imaging in the scanning transmission electron microscope (STEM) provides an image with strong atom-number contrast and is sufficient to distinguish between different cell types and different organelles within the cells of the DRG. We also show that Pt may be imaged at the single atom level and be localised at very high resolution while still preserving a degree of ultrastructural context. The intrinsic image contrast generated is sufficient to identify these features without the need for heavy metal stains and other extensive processing steps which risk disturbing native platinum distributions within the tissue. We subsequently demonstrate that by considering the total elastic scattering intensity generated by nanometre-sized Pt aggregations within the cell, the ADF STEM may be used to make a measurement of local concentration of Pt in units of atoms per cubic nanometre. We further estimate the minimum atomic number required to visualise single atoms in this setting, concluding that in similar samples it may be possible to detect species as light as sodium with atomic sensitivity.

Keywords: dorsal root ganglia; high‐angle annular dark field imaging; neuron; oxaliplatin; scanning transmission electron microscopy; single atom imaging.

FIGURE 1

Low‐magnification ADF images recorded from a thin section of DRG allows for identification of regions of interest. The sample exhibits good ultrastructural preservation. (A) This image facilitate the detection of sensory neuronal cell bodies (α, β) and myelinated axons (γ). (B) A higher magnification allows observation of neuronal cell bodies (α), nuclear membranes (β), nucleoli (γ), small myelinated axons (δ), capillaries (ε) and other cells within the DRG (ζ).

FIGURE 2

(A) Higher‐magnification ADF images show identifiable subcellular features such as mitochondria (M), the nucleolus (Nu) and nuclear membrane (NM). (B) Some regions in the DRG cytosol exhibit increased image contrast, correlating to higher average atomic number or density in that region. The inset shows an example of such a region on the exterior of the nuclear membrane which was subsequently found to contain Pt. The electron dose used to acquire images of the type displayed in (B) was on the order of 20–200 electrons per Ų.

FIGURE 3

EDX spectrum acquired from Pt‐containing clusters in DRG. (A) Features similar to those in Figure 2 are visible throughout the DRG. (B) EDX acquired from continuously scanning over the inset region shows characteristic Pt x‐rays appearing as shoulders on the nearby P Kα and S Kα peaks. Due to limitations on electron dose, these peaks are at most only slightly above the spectral noise level. Qualitatively, the spectrum shows the sample is mostly comprised of carbon, nitrogen, oxygen, with trace amounts of other elements.

FIGURE 4

ADF images of atomic clusters located using high‐magnification STEM. Single atoms are identified by arrowheads.

FIGURE 5

Image intensity line profile demonstrating the ångström (Å)‐scale of the single‐atom peaks.

FIGURE 6

(A) Response map of the upper ADF detector in the Oxford JEOL ARM‐200F. Non‐uniform intensity indicates the response is not homogeneous across the detector. (B) Atomic cluster image converted to units of fractional beam intensity. The most intense areas indicate as much as 12% of the beam is scattered to the ADF detector at some probe positions.

FIGURE 7

Logarithmic plot of simulated ADF scattering cross‐section against atomic number. The inset graph shows expected cross‐sections for iridium, platinum and gold (Z = 77, 78 and 79, respectively). Experimental data with an error bar of one standard deviation are also shown in black.

FIGURE 8

Atomic cluster weighing in the DRG. Experimental data points are fitted with a cubic dependence upon cluster diameter (solid line). The results of this fit indicate that Pt clusters found within the DRG contain approximately 40% fewer atoms per cubic nanometre when compared to an equivalently sized metallic Pt nanoparticle (dotted line). Within the DRG, the average cluster diameter is around 3.5 nm, and contains approximately 1000 atoms.

FIGURE 9

Minimum detectable atomic number of single atoms embedded in a low‐Z matrix. Detectability is strongly dependent upon electron fluence, but also upon sample thickness and image integration area.