Electron spectroscopic imaging for high-resolution immunocytochemistry: use of boronated protein A.

Abstract

In the present study we adapted electron spectroscopic imaging (ESI) for high-resolution immunocytochemistry. To accomplish this, we applied boronated protein A (B-pA) for indirect detection of specific antigenic sites using pre-embedding and post-embedding protocols. Isolated acinar cells were exposed to wheat germ agglutinin (WGA) and anti-WGA, followed by B-pA, to reveal WGA binding sites at the level of the plasma membrane. The cells were then embedded in Epon and unstained ultra-thin sections were examined by electron microscopy using the ESI mode. For post-embedding, ultra-thin sections of glutaraldehyde-fixed, Lowicryl-embedded pancreatic tissue were exposed to specific antibodies (anti-insulin or anti-amylase), followed by B-pA. The unstained sections were examined using the ESI mode. In both cases, boron was detected with high resolution either at the level of the plasma membrane of acinar cells, demonstrating WGA binding sites, or over secretory granules in pancreatic insulin-secreting cells or acinar cells, demonstrating insulin and amylase, respectively. These findings were compared to those obtained with the protein A-gold technique, and have demonstrated the analogy of both types of labeling. In addition, several control experiments assessed this novel approach. They have demonstrated the specificity of labeling and the high reactivity of B-pA, as well as its antibody-binding properties. Finally, electron energy loss spectral analysis confirmed the presence of boron in the tissue sections at sites where immunolabeling was detected. These results demonstrate that ESI is an appropriate approach for cytochemistry. Since the technique is based on detection of elements, spatial resolution is considered to be in the magnitude of 0.5 nm, which represents a major improvement in resolution over actual electron microscopic cytochemical techniques.

Calculation of boron neutron capture cell inactivation in vitro based on particle track structure and x-ray sensitivity

“…These restrictions can be avoided in future developments of the model if the real microdistribution of 10 B and the complete cell size distribution of asynchronous cell populations are taken into account. Techniques used for the quantitative determination of microdistributions of boron compounds in several tumor cell lines or in samples of glioblastoma include electron spectroscopic imaging/energy loss spectroscopy (ESI/EELS) [40,41], secondary ion mass spectroscopy (SIMS) [42], laser microprobe mass analysis (LAMMA) [43], and high resolution alpha-track autoradiography [44]. A more realistic model for microdosimetry may be constructed using dynamic modeling, which offers computer simulations of internal cell structures with variable geometry of the cells dependent on the cell cycle stage.…”

Synthesis of New Boron‐Rich Building Blocks for Boron Neutron Capture Therapy or Energy‐Filtering Transmission Electron Microscopy

“…Previous studies [2,6,18] of boron imaging in cells or tissues have been carried out by using EFTEM. A spherical shape of the molecule carrying boron and the presence of a relatively high number of boron atoms in a small volume improves the detectability in EFTEM.…”

Immunocytochemistry by electron spectroscopic imaging using a homogeneously boronated peptide

“…Electron spectroscopic imaging (ESI; Ottensmeyer and Andrew 1980;Ottensmeyer 1984;Colliex 1986) allows for spatial resolutions of 3-5 Å, provided that the detection limit of some 50 compounded atoms is met or exceeded, as shown for phosphorous by Adamson-Sharpe and Ottensmeyer (1981). Bendayan et al (1989) were the first to apply the ESI technique to immunocytochemistry. They covalently linked several carborane clusters to protein A to identify cellular antigens complexed with appropiate antibodies.…”