Quantitative DNA measurements in an instrument combining scanning electron microscopy and light microscopy

Abstract

SUMMARY An instrument for combined scanning electron microscopy (SEM) and light microscopy (LM) to which a photometer unit is attached is described. A special stage in the vacuum chamber of a scanning electron microscope incorporates light microscope optics (objective and condenser) designed for transmission and epi‐illumination fluorescence LM. An optical bridge connects these optics to a light microscope, without objective and condenser. The possibility of performing quantitative DNA measurements in this combined microscope (the LM/SEM) was tested using preparations of either chicken erythrocytes, human lymphocytes, or mouse liver cells. The cells were fixed, brought on a cover‐glass, quantitatively stained for DNA, dehydrated, and critical point dried (CPD). After mounting the cells were coated with gold. The specimens were brought into the vacuum chamber of the combined microscope and individual cells were studied with SEM and LM. Simultaneously DNA measurements were performed by means of the photometer unit attached to the microscope. It is shown in this study that DNA measurements of cells in the combined microscope give similar results when compared to DNA measurements of embedded cells performed with a conventional fluorescence microscope. Furthermore, it is shown that although the gold layer covering the LM/SEM specimens weakens the fluorescence signal, it does not interfere with the DNA measurements.

A simple photographic grid system for the correlative examination of cell smears by light and scanning electron microscopy

“…A method for examining cells smeared on to 'slide-glassed-sized X-ray film' by LM and SEM has been described (It0 81 Kudo, 1982) but the authors did not elaborate on how they achieved precise identification of cells seen at LM and later SEM. More recently a specially developed instrument consisting of a scanning electronmicroscope with light microscope in tandem along with an attached photometer unit has been described (Wouters et al, 1986). This instrument allows the same cells to be examined by LM and SEM with great precision.…”

Fluorescence CLEM in biology: historic developments and current super‐resolution applications

“…The first examples of studies that truly exploit CLEM to derive different types of biological information from the same sample include work on spreading macrophages [4] and white blood cells [5]. In spreading macrophages, FM was used to demonstrate the existence of actin-rich structures while the EM was used to resolve actin fibres that connect these structures, something which could not be resolved by FM alone at the time [4].…”

Techniques for combining light microscopy and scanning electron microscopy: a survey of the literature

“…Immunocytochemistry is possible, however, with some limitations. The attachment of a photometer unit (Leitz, MPV-11, West Germany) makes quantitative DNA measurements of appropriately stained cells possible (Wouters et al, 1986).…”