A Method for Obtaining Serial Ultrathin Sections of Microorganisms in Transmission Electron Microscopy

Summary

This study presents reliable and easy procedures for obtaining serial ultrathin sections of a microorganism without expensive equipment in transmission electron microscopy.

Abstract

Observing cells and cell components in three dimensions at high magnification in transmission electron microscopy requires preparing serial ultrathin sections of the specimen. Although preparing serial ultrathin sections is considered to be very difficult, it is rather easy if the proper method is used. In this paper, we show a step-by-step procedure for safely obtaining serial ultrathin sections of microorganisms. The key points of this method are: 1) to use the large part of the specimen and adjust the specimen surface and knife edge so that they are parallel to each other; 2) to cut serial sections in groups and avoid difficulty in separating sections using a pair of hair strands when retrieving a group of serial sections onto the slit grids; 3) to use a 'Section-holding loop' and avoid mixing up the order of the section groups; 4) to use a 'Water-surface-raising loop' and make sure the sections are positioned on the apex of the water and that they touch the grid first, in order to place them in the desired position on the grids; 5) to use the support film on an aluminum rack and make it easier to recover the sections on the grids and to avoid wrinkling of the support film; and 6) to use a staining tube and avoid accidentally breaking the support films with tweezers. This new method enables obtaining serial ultrathin sections without difficulty. The method makes it possible to analyze cell structures of microorganisms at high resolution in 3D, which cannot be achieved by using the automatic tape-collecting ultramicrotome method and serial block-face or focused ion beam scanning electron microscopy.

Introduction

Proper serial ultrathin sectioning technique is indispensable to study cells and cell components three-dimensionally at the electron microscopic level. We have studied the dynamics of spindle pole body in the cell cycle of yeast cells, and revealed morphological changes of their ultrastructure during the cell cycle and the time of duplication^1,2,3,4,5. In 2006, we coined a new word 'structome' by combining 'structure' and '-ome', and defined it as the 'quantitative and three-dime....

Protocol

NOTE: The specimens used in this study were microorganisms, rapidly frozen with propane in liquid nitrogen, freeze substituted in acetone containing 2% osmium tetroxide, and embedded in epoxy resin^{1,2,3,4,5,6,7,8,9,10,11,12,13,....}

Results

In this protocol, three-slit grids were used for picking up serial sections. The grids are made of nickel or copper. The serial sections are placed on the middle slit. The slits on both sides are necessary to view the sections when picking them up with the grid. To keep the grids parallel with the serial sections when picking them up with tweezers (Figure 11d), the handle is bent (Figure 6c, right). A small handle is advantageous.......

Discussion

The method presented here requires no expensive equipment. It requires only an aluminum rack (Figure 3), three-slit grids (Figure 6c), section-holding loops (Figure 10a), water-surface-raising loop (Figure 11a), and a staining tube (Figure 13). There are many features of the present method. The large part of the specimen is used to adjust the specimen surface and knife edge.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Formvar making apparatus	Nisshin EM Co. Ltd., Tokyo	652	W 180 x D 180 x H 300 mm
Glass slide	Matsunami Co. Ltd., Osaka	-	76 x 26 x 1.3 mm
Aluminum rack with 4-mm holes	Nisshin EM Co. Ltd., Tokyo	658	W 30 x D 25 x H 3 mm, Refer to this paper
Stereomicroscope	Nikon Co. Ltd., Tokyo	-	SMZ 645
LED illumination for stereomicroscope	Nikon Co. Ltd., Tokyo	-	SM-LW 61 Ji
Trimming stage	Sunmag Co.Ltd., Tokyo	-	Tilting mechanism equipped, Refer to this paper
LED illumination for trimming stage	Sunmag Co.Ltd., Tokyo	-	Refer to this paper
Ultrasonic trimming blade	Nisshin EM Co. Ltd., Tokyo	5240	EM-240, Refer to this paper
Diamond knife for trimming	Diatome Co. Ltd., Switzerland	-	45°
Diamond knife for ultrathin sectioning	Diatome Co. Ltd., Switzerland	-	45°
Ultramicrotome	Leica Microsystems, Vienna	-	Ultracut S
Mesa cut	Leica Microsystems, Vienna	-	Mirror
0.5% Neoprene W solution	Nisshin EM Co. Ltd., Tokyo	605
Special 3-slit nickel grid	Nisshin EM Co. Ltd., Tokyo	2458	Refer to this paper
Special 3-slit copper grid	Nisshin EM Co. Ltd., Tokyo	2459	Refer to this paper
Section-holding loop	Nisshin EM Co. Ltd., Tokyo	526	Refer to this paper
Water-surface-raising loop	Nisshin EM Co. Ltd., Tokyo	527	Refer to this paper
Staining tube	Nisshin EM Co. Ltd., Tokyo	463	Refer to this paper
Multi-specimen holder	JEOL Co. Ltd., Tokyo	-	EM-11170
JEM-1400	JEOL Co. Ltd., Tokyo	-	Transmission electron microscope