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SEM Pathology

Table of contents

3. Advantages of LVSEMs and future tasks

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1. Examination of common tissue samples #000001

Sample preparation and staining

A tabletop SEM allows for the observation of “paraffin sections” prepared for light microscopy3),4).
After initial treatment with a commonly used fixative (such as 10% formalin or 4% paraformaldehyde), tissue samples are dehydrated and embedded in paraffin. These paraffin blocks are then cut into thin sections (5–10 μm in thickness), mounted on standard-size glass slides, and pretreated as described below. Even after washing with water, sections that are slightly wet can be placed in the sample chamber of a LVSEM for observation.

Sample preparation method

Deparaffinization100% xylene (3 times) → 100–50% ethanol → Distilled water (When examining samples previously used for light microscopy, the coverslips must be removed prior to reaching the steps utilizing 100–50% ethanol and distilled water. This can be achieved by completely immersing the slide in an organic solvent, such as xylene.)
StainingSelect an appropriate stain based on the research objectives.
(a) Platinum blue: adjust the pH of the staining solution included in the TI-Blue kit (Nisshin EM Co., Ltd.) to 9, apply one drop of the solution to each tissue section, and incubate at room temperature for 10–20 min.
(b) Other heavy-metal staining agents (such as osmium tetroxide)
WashingWash with distilled water for 1–2 min. (Absorb excess water around the tissue section using a filter paper, for example. Do not use a coverslip.)Apply double-adhesive carbon tape to the center of an SEM sample stub and firmly attach a glass slide to the tape with the tissue section positioned at the center of the stub.
Observation with a LVSEMAccelerating voltage, 15 kV; vacuum, 30 – 50 Pa; backscattered electron images


Tabletop SEMs can be effectively operated under low-vacuum conditions, thereby generally eliminating the need for extensive sample preparation, such as pretreatment or metal coating. However, the thorough observation of biological specimens requires heavy-metal staining. Such staining enhances backscattered electron signals and allows for histologically specific staining. The specificity results from differences in the staining intensity of cellular and tissue components and leads to improved identification of various types of cells and tissues.

Staining with “platinum blue” or “osmium tetroxide” is effective for examining standard animal tissue samples. Here, we present several studies that utilized these stains for immunohistochemistry.

(a) Staining properties of platinum blue

Platinum blue staining was developed in 1993. It is a staining method designed to increase the backscattered electron signals from biological samples1). Staining with platinum blue is a very simple process, requiring only the immersion of samples for 10–20 min in stain solution adjusted to pH 9. Stained tissue specimens appear blue; thus, they can be examined using a light microscope. Platinum blue also provides histologically specific staining (due to differences in the staining intensity of cellular and tissue components). This results in a contrast in brightness under a LVSEM and facilitates the detection of different types of cells and tissues. Additionally, with higher magnifications, LVSEMs allow for further characterization of these cells and tissues and even enable three-dimensional analysis of structures.

(b) Staining properties of osmium tetroxide

In addition to platinum, tissue-specific staining can be performed using other heavy metals that provide a strong contrast in LVSEM images. The heavy metal compound osmium tetroxide (OsO4) is frequently used in the preparation (e.g., fixing and conductive staining) of samples for general electron microscopy. This compound is also used for intensifying DAB color development in immunohistochemical staining of light microscopy samples. Tissue areas positive for the OsO4 reaction produce a strong contrast against the surrounding tissue. Thus, this heavy-metal stain is applicable even in experiments using LVSEMs. However, OsO4 must be handled with great caution due to its high toxicity.

Here, we describe a representative study that examined pathologic tissue samples of breast cancer (invasive ductal carcinoma) using a LVSEM.

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