Sample Preparation Method of Scanning and Transmission Electron Microscope for the Appendages of Woodboring Beetle

Summary

In order to observe ultrastructure of insect sensilla, scanning and transmission electron microscopy (SEM and TEM, respectively) sample preparation protocol were presented in the study. Tween 20 was added into the fixative to avoid sample deformation in SEM. Fluorescence microscopy was helpful for improving slicing accuracy in TEM.

Abstract

This report described sample preparation methods that scanning and transmission electron microscope observations, demonstrated by preparing appendages of the woodboring beetle, Chlorophorus caragana Xie & Wang (2012), for both types of electron microscopy. The scanning electron microscopy (SEM) sample preparation protocol was based on sample chemical fixation, dehydration in a series of ethanol baths, drying, and sputter-coating. By adding Tween 20 (Polyoxyethylene sorbitan laurate) to the fixative and the wash solution, the insect body surface of woodboring beetle was washed more cleanly in SEM. This study's transmission electron microscopy (TEM) sample preparation involved a series of steps including fixation, ethanol dehydration, embedding in resin, positioning using fluorescence microscopy, sectioning, and staining. Fixative with Tween 20 enabled penetrate the insect body wall of woodboring beetle more easily than it would had been without Tween 20, and subsequently better fixed tissues and organs in the body, thus yielded clear transmission electron microscope observations of insect sensilla ultrastructures. The next step of this preparation was determining the positions of insect sensilla in the sample embedded in the resin block by using fluorescence microscopy to increase the precision of target sensilla positioning. This improved slicing accuracy.

Introduction

Scanning electron microscopy is an important tool in many morphology studies, that SEM shows surface structures^1,2. Transmission electron microscopy's appeal is that it can be used to study a wide range of biological structures at the nanometer scale, from the architecture of cells and the ultrastructure of organelles, to the structure of macromolecular complexes and proteins. TEM shows inner structures^3,4,5.

Coleoptera is the largest group of insects, including about 182 families and 3....

Protocol

CAUTION: Consult the material safety data sheets of reagents before using them. Several of the chemicals used during sample preparation are toxic, mutagenic, carcinogenic, and/or reprotoxic. Use personal protective equipment (gloves, lab coat, full-length pants, and closed-toe shoes) and work under a fume hood while handling the sample.

1. SEM Sample Preparation and Imaging

1. Sample Fixation and Cleaning
   1. Working in an area where C. caragana occur, attract adults into field traps baited with plant attractants, such as isophorone¹⁶. Preserve clean bodies of adult C. caragana in 0....

Results

Using cleaning and fixative solution with Tween 20, cleaner SEM image was observed than that without Tween 20 (Figure 3). Tween 20 fixing solution penetrated the glutaraldehyde fixing solution into the tissue. Microtubule structure was clearly seen. TEM image of the internal structure of the sample was blurred without Tween 20 (Figure 4).

Discussion

In this article, we presented a sample preparation scheme for scanning and transmission electron microscopy for woodboring beetle. Using insect appendage as a representative study subject, we demonstrated several improvements over traditional sample preparation methods.

The liquid oil detached from the solid surface is emulsified into small droplets, which can be well dispersed and suspended in the washing medium to reduce redepositing on the surface of the object. Washing performance of surfa.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Anatomical lens	Chongqing Auto Optical limited liability company	1425277
Carbon adhesive tape	SPI Supplies, Division of Structure Probes, Inc.	7311
Carbon tetrachloride	Sigma	56-23-5
Copper grids	GilderGrids	G300
Disodium hydrogen phosphate	Sinopharm group chemical reagent co., LTD	10039-32-4
Ethanol	J.T. Baker	64-17-5
Flat embedding molds	Hyde Venture (Beijing) Biotechnology Co., Ltd.	70900
Fluorescence microscope	LEICA	DM2500
Glutaraldehyde	Sigma-Aldrich	111-30-8	Anhydrous EM Grade
Isophorone	Sigma	78-59-1
Lead citrate	Sigma	512-26-5
Methanol	Sigma	67-56-1
Monobasic sodium phosphate	Its group chemical reagent co., LTD	7558-80-7
Objective micrometer	Olympus	0-001-034
Osmium tetroxide	Sigma	541-09-3
Petri dish	Aldrich	1998
Razor blade	Gillette
Resin	Spurr	ERL4221
Scalpel	Lianhui	GB/T19001-2008
SEM	Hitachi	S-3400
Silica gel desiccant	Suzhou Longhui Desiccant Co., Ltd.	112926-00-8
Small brush	Martol	G1220
Sodium hydroxide	Sigma	1310-73-2
Sputter ion instrument	Hitachi Koki Co. Ltd., Tokyo, Japan	E-1010
Stereo microscope	Leica	EZ4 HD
TEM	Hitachi	H-7500
Tween 20	Tianjin Damao Chemical Reagent	9005-64-5
Ultramicrotome	Leica	UC6
Ultrasonic cleaner	GT Sonic	GT-X1
Uranyl acetate	Sigma	6159-44-0