FIB-SEM imaging of carbon nanotubes in mouse lung tissue

Abstract

Ultrastructural characterisation is important for understanding carbon nanotube (CNT) toxicity and how the CNTs interact with cells and tissues. The standard method for this involves using transmission electron microscopy (TEM). However, in particular, the sample preparation, using a microtome to cut thin sample sections for TEM, can be challenging for investigation of regions with agglomerations of large and stiff CNTs because the CNTs cut with difficulty. As a consequence, the sectioning diamond knife may be damaged and the uncut CNTs are left protruding from the embedded block surface excluding them from TEM analysis. To provide an alternative to ultramicrotomy and subsequent TEM imaging, we studied focused ion beam scanning electron microscopy (FIB-SEM) of CNTs in the lungs of mice, and we evaluated the applicability of the method compared to TEM. FIB-SEM can provide serial section volume imaging not easily obtained with TEM, but it is time-consuming to locate CNTs in the tissue. We demonstrate that protruding CNTs after ultramicrotomy can be used to locate the region of interest, and we present FIB-SEM images of CNTs in lung tissue. FIB-SEM imaging was applied to lung tissue from mice which had been intratracheally instilled with two different multiwalled CNTs; one being short and thin, and the other longer and thicker. FIB-SEM was found to be most suitable for detection of the large CNTs (Ø ca. 70 nm), and to be well suited for studying CNT agglomerates in biological samples which is challenging using standard TEM techniques.

Figure

3D FIB-SEM image reconstruction of carbon nanotube (CNT) sample in lung tissue obtained with the double tilted milling method. A few CNTs have manually been traced in the 3D volume, and the white arrowheads point to a single CNT. A – alveole, E – erythrocyte, and P1 – pneumocyte (type 1).

Fig. 1

TEM micrographs of the two CNT types used. a CNT_Small. b CNT_Large

Fig. 2

Schematic of the milling geometries used. a Standard milling approach where the sample is tilted 52°. b The double non-tilted milling method, where the milling is performed without tilting the sample and a wedge is created which has two FIB polished surfaces

Fig. 3

TEM micrographs of lung tissue with CNT_Small (a–b) and CNT_Large (c–d). a Overview image of a large agglomeration of CNT_Small in the region where black arrowheads highlight very dense CNT agglomerates in the alveolar lumen. The CNTs are causing minor microtomy artefacts (stripes extending from the middle of the image towards the lower right corner). b A CNT_Small is seen interacting with a cell (insert), and a CNT observed freely inside the cytosol (white arrow). c Overview image showing CNT_Large between cells causing major microtomy artefacts. d TEM image of a dense agglomeration of CNT_Large resulting in holes and stripes in the ultrasection. A alveoli, AM Alveolar macrophage, E erythrocyte, N nucleus, P1 pneumocyte (type 1), and P2 pneumocyte (type 2). Black arrowheads indicate CNT agglomerates, whereas white arrowheads indicate single CNTs

Fig. 4

SEM micrographs of the exposed block surface following ultramicrotomy. a Image of the CNT_Small sample protruding slightly from the microtomed surface. b A CNT_Large sample after microtomy, where large protruding CNTs are seen which in some cases have left an impression in the Epon block during flattening. c Low magnification overview of how lung tissue in close proximity to CNTs can be visualised and later targeted with FIB-SEM

Fig. 5

FIB-SEM micrographs of both types of CNTs in the lung samples. a–b The CNT_Small sample imaged with standard milling including a platinum layer, where it can be difficult to discern CNTs from cellular material. Black arrowheads mark the likely agglomerations of CNTs not observed in control samples and correlated with CNTs protruding from the surface of the Epon block. One cell appears to have a large invagination, possibly containing CNTs (small white arrowheads). c–d CNT_Large samples obtained via standard milling, but without protective platinum layer. Here, the milling artefacts (vertical white lines) caused by surface roughness is clearly seen (especially in c). However, the cells and CNTs are still visible, and single CNTs can be found to interact closely with the tissue, but are only in very few cases observed to appear entering the alveolar wall (white arrowheads in d). e–f FIB-SEM of CNT_Large using the double non-tilted milling approach limiting surface roughness caused artefacts, with arrowheads highlighting the protruding CNTs caused by differing milling yields. f SEM image obtained from the viewpoint of the ion beam, showcasing that CNTs protrude from the milled surface. A alveoli, E erythrocyte, L lamella body, M mitochondrion, N nucleus, and P2 pneumocyte (type 2)

Fig. 6

3D FIB-SEM image reconstruction of CNT_Large sample obtained with the double non-tilted milling method. a Orthogonal xy, xz and yz-views of the stack. b 3D view with semi-transparent rendering of the stack. To illustrate the possibility of manually tracing CNTs in 3D a few of the CNTs have been manually coloured blue in Amira. The white arrows point to the same CNT in both views. A alveoli, E erythrocyte, and P1 pneumocyte (type 1)

Fig. 7

Comparison of the resolution obtainable with TEM and FIB-SEM images of CNTs in lung tissue. a–b TEM micrographs of the CNT_Small and CNT_Large sample, respectively. The CNTs can be distinguished from cellular material. c–d FIB-SEM equivalents of the CNT_Small and CNT_Large sample, respectively. The micrographs were obtained with the standard milling method including the platinum layer. CNT_Large can be visualised as two parallel lines which depending on the imaging method have a weak signal from the centre. But as we approach the resolution limitations of the FIB-SEM the small CNTs are simple lines undistinguishable from cellular material. White arrowheads denote single CNTs, and black arrowheads agglomerates of CNTs