Elsevier

Micron

Volume 43, Issue 5, April 2012, Pages 612-620
Micron

Beam deceleration for block-face scanning electron microscopy of embedded biological tissue

https://doi.org/10.1016/j.micron.2011.11.001Get rights and content

Abstract

The beam deceleration (BD) method for scanning electron microscopes (SEM) also referred to as “retarding” was applied to back-scattered electron (BSE) imaging of the flat block face of a resin embedded biological specimen under low accelerating voltage and low beam current conditions. BSE imaging was performed with 0–4 kV of BD on en bloc stained rat hepatocyte. BD drastically enhanced the compositional contrast of the specimen and also improved the resolution at low landing energy levels (1.5–3 keV) and a low beam current (10 pA). These effects also functioned in long working distance observation, however, stage tilting caused uncorrectable astigmatism in BD observation. Stage tilting is mechanically required for a FIB/SEM, so we designed a novel specimen holder to minimize the unfavorable tilting effect. The FIB/SEM 3D reconstruction using the new holder showed a reasonable contrast and resolution high enough to analyze individual cell organelles and also the mitochondrial cristae structures (∼5 nm) of the hepatocyte. These results indicate the advantages of BD for block face imaging of biological materials such as cells and tissues under low-voltage and low beam current conditions.

Highlights

► Beam deceleration (BD) applied to a block face SEM imaging (BFI) of embedded cell. ► BD drastically enhanced the contrast and resolution (∼5 nm) of the BFI of a cell. ► Effect remarkable at low accelerate voltage (1.5 kV) and low beam current (10 pA). ► Newly developed device allows 3D imaging using FIB/SEM with BD. ► BD improved 3D reconstruction allows mitochondrial cristae structural analysis.

Introduction

Backscattered electron (BSE) imaging with the scanning electron microscope (SEM) from the flat cut surface of resin embedded biological materials provides correlative images from transmission electron microscopy (TEM) without ultrathin sectioning (Richards, 1996, Denk and Horstmann, 2004). This block face image (BFI) is able to provide the structural information of the specimen at a higher resolution than light microscopy (LM) and over a wider area than the TEM. The recent three-dimensional (3D) reconstruction methods known as serial block face SEM (SBFSEM) (Denk and Horstmann, 2004) and focused ion beam scanning electron microscope (FIB/SEM) tomography (Knott et al., 2008, Kato et al., 2007, De Winter et al., 2009) provide one of the most successful applications for biological specimens. However, the signal-noise ratio of the BFI is generally lower than in the TEM imaging (Jurrus et al., 2009).

Compositional contrast is known to be the principle of block face imaging. When the primary electron beam scans a flat and smooth surface made by a diamond knife on an ultramicrotome of a resin block of heavy metal-stained biological material, the emitted BSE signal does not contain topological information, but the contrast depends on the atomic composition of specimen (Richards, 1996, Richards and Gwynn, 1995). Materials with larger atomic numbers such as heavy metals provide a higher probability of BSE emission (Assa’d and El Gomati, 1998) and accordingly, the BFI displays a “brighter” intensity for heavy metal stained objects such as biological membranes stained with osmium and uranium, and “darker” for light elements such as the surrounding embedding resin.

For SEM in principle, the accelerating voltage of the primary electron correlates with the penetration and interacting volume of the electron, hence the final resolution of images. For higher resolution, low accelerating voltage imaging is required to acquire compositional information from the very near surface of the specimen (Denk and Horstmann, 2004). However, in BFIs obtained under low accelerating voltage, signal-noise ratio is low and the image contrast is not sufficient especially when using a commonly used SEM that places the specimen below the lens (conventional SEM). Consequently, a favorable imaging of the BFI needs some signal enhancement as follows: (1) usage of SEM system designed for low-voltage observation such as a semi-in-lens SEM; (2) multiple en bloc staining of the specimens to increase the number of BSEs (Deerinck et al., 2010); (3) a high sensitive detector optimized for low voltage BSE (Denk and Horstmann, 2004); (4) a short working distance (WD) between the pole piece and the specimen; (5) a large beam current (probe current) for imaging to increase the number of BSEs (Bushby et al., 2011); (6) digital image processing after image acquisition(Jurrus et al., 2009). A combination of procedures (1)–(4) improves the image quality. In procedure (5) the large beam current reduces the image resolution, but increases the signal amount. Therefore, a more sensitive detection method is required to obtain sufficient contrast when using low acceleration voltage and low beam current.

A beam deceleration (BD) method, also referred to as “retarding”, has been known to improve the resolution and contrast of BSE imaging in material science research (Phifer et al., 2009a). This method is a simple way to reduce electron beam energy at a cathode bias voltage of 1–4 kV between the specimen and the SEM pole piece. The original motivation for the BD method was to achieve reasonable imaging at very low energy down to a few hundred electron volts to observe the surface structure of the specimen (Müllerová and Frank, 2003). This cathode bias voltage, which forms an electro static lens (cathode lens) between the sample and pole piece, affects the optical properties of the electron and improves SEM imaging at low accelerating voltage.

The BD method has not been applied to the BFI of biological specimens or with the recent 3D reconstruction methods using BFI. Thus the effect of the BD on a BFI of the biological specimen has not been well understood, although BD has been used for biological applications to observe the surface of cultured cells under very low voltage conditions (Pluk et al., 2009). In the work presented here, we applied and evaluated the effect of BD for the BFI of embedded biological specimens and the influence of accelerating voltage, WD, and stage tilting under a low beam current with BD conditions using a conventional field emission SEM.

Upon the usage of BD, the large protrusion and tilting of the specimen, which are unavoidable for micro fabrication and observation using a FIB/SEM, disturb the symmetry cathode lens and cause distortion and astigmatism (Phifer et al., 2009b). So we propose a novel specimen holder to solve the problem and we applied it to the 3D reconstruction method using a FIB/SEM (Knott et al., 2008), which rebuilds the 3D structure of the specimen by a repeat cycle of ion-beam milling and BFI acquisition of the specimen.

Section snippets

Preparation of biological tissue specimen

Rat (Wistar) liver tissue was used for a representative example of a biological specimen. The specimens were obtained in accordance with the guidelines established by the Institutional Animal Committee of Kurume University School of Medicine. The animals were deeply anesthetized with diethyl ether and sodium pentobarbital (50 mg/kg), and were transcardially perfused through the left ventricle with heparin containing (10 U/ml) saline, followed by fixative as 2% paraformaldehyde, 2.5%

Effect of the beam deceleration for block face imaging

The application of BD dramatically increased the compositional contrast of the image taken from the flat surface of the specimen and displayed the structure detail of the cut surface of resin embedded liver tissue in both low and high magnification (Fig. 2). The raw BFIs looked like negative images of the TEM micrographs (Fig. 2a, b, e and f). Thus, contrast-inverted images correspond to the TEM images (Fig. 2c and g). When the primary electron beam scanned a rough surface such as a surface

Discussion

In this study, we have shown that BD drastically improves the compositional contrast in block face imaging of embedded biological specimens viewed under a conventional SEM. Although we used a conventional type FE-SEM based instrument for the imaging, the contrast is sufficiently high to render unnecessary any post digital image processing. The improvement in signal detection efficiency is remarkable at low accelerating voltage and low beam current conditions. This is indispensable in order to

Conclusion

Beam deceleration is beneficial for block face imaging of resin embedded biological specimens especially at low-voltage and low beam current conditions, which improve the image contrast and resolution. The lateral resolution reaches approximately 5 nm and the contrast is sufficiently high without any additional digital processing. This quality corresponds to a conventional TEM image for the purpose of general biological observations. Furthermore, a combination of an optimized microscope setting

Author contributions

K.O. and K.N. designed the research. A.T. and R.T prepared the specimens. S.S. and R.H. performed the FIB/SEM analysis. K.O. co-write the manuscript.

Acknowledgement

We thank P. Debbie for providing language help and reading the manuscript.

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