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Overview

SEM Pathology

Table of contents

1. Examination of common tissue samples

Staining properties of platinum blue

Staining properties of osmium tetroxide

2. Examination of renal biopsy samples

3. Advantages of LVSEMs and future tasks

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Staining properties of osmium tetroxide　#000006

(2) Immunohistochemical staining of breast cancer tissue samples (LVSEM images)

Figure No.000006a

Staining: DAB color development only

Microscope: LVSEM
Sample: Paraffin section

Accelerating voltage: 15 kV
Magnification: ×1,500

Image description
LVSEM image of a DAB-positive area (infiltrating lactiferous duct). Cell morphology is unclear.

Figure No.000006b

Staining: DAB color development + Osmium tetroxide staining

Microscope: LVSEM
Sample: Paraffin section

Accelerating voltage: 15 kV

Magnification: ×1,200

Image description
OsO4 intensification applied after DAB color development increases image contrast. The entire ductal epithelium turns bright, and the morphology of nucleus (→), are clearly discernable.

Figure No.000006c

Staining: DAB color development only

Microscope: LVSEM
Sample: Paraffin section

Accelerating voltage: 15 kV

Magnification: ×4,000

Image description
When tissue sections are stained with DAB alone, cell morphology remains unclear even at higher magnifications.

Figure No.000006d

Staining: DAB color development + Osmium tetroxide staining

Microscope: LVSEM
Sample: Paraffin section

Accelerating voltage: 15 kV

Magnification: ×2,500

Image description
Following OsO4 intensification, DAB-positive areas (i.e., ductal epithelia) become distinctly bright. At higher magnifications, the morphology of cells and subcellular structures, such as the nucleus (→), can be clearly discernable.

Characteristic findings

When breast cancer tissue is stained with DAB alone, the morphology of the cells in DAB-positive areas remains indiscernible under a LVSEM. However, on OsO4 intensification, DAB-positive cells provide a strong contrast against the surrounding tissue, making it possible to clearly capture the details of their microstructure. At higher magnifications, LVSEMs also allow for the examination of the detailed morphology of these cells and their organelles (such as the nucleus).11)

Observations

Samples shown in Fig. 000006 (panels a and c) were stained with DAB alone, while those in panels b and d were treated with 2% OsO4 for one hour for color intensification after DAB staining. All samples were examined with a LVSEM. Color development with DAB alone fails to precisely delineate DAB-positive areas (Figs. 000006 a and c). However, OsO4 intensification enhances the contrast of these areas and provides well-defined images at higher magnifications.

Reference

11) Inaga S, et al. The 119th Annual Meeting of the Japanese Association of Anatomists (March 27 – 29, 2014: Jichi Medical University)

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