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 350,000 species. Most of the coleopteran insects, in particular woodboring beetle, feed on plants, many of which are important pests of forests and fruit trees, causing devastating damage to trees⁶. At present, prevention and control population of pests based on chemical ecology theory have received increasing attention⁷. Efficient, low-toxic, pollution-free pheromone control methods have become an effective way⁸. Studying the sensilla morphology and ultrastructure of insects is an important part of insect chemical ecology research. The scanning and transmission electron microscopy (SEM and TEM, respectively) are used to great effect to study their morphology and internal anatomy. However, during preparation of insect samples for electron microscopy (EM), the objectivity and authenticity of the observation site may be affected⁹. In general, SEM sample preparation of insects requires cleaning, tissue fixation, dehydration, metathesis, drying, and sputter-coating¹⁰. Due to the complex environment in which woodboring beetle live, the body surface often has various pollutants and their appendages often have many fine long sensilla or bristles. In particular, some woodborers are not available from laboratory raising, which collected directly in the field, and then put into fixing fluid to ensure freshness and subsequently washed in the laboratory. If the sample is first fixed and then washed, obviously it is much more difficult to remove debris because glutaraldehyde strongly fixes it to the sample. Tween 20 is a surfactant^11,12,13,14, which plays an important role in the washing process, including reducing the surface tension of water and improving the wettability of water on the surface of the laundry. In this study, Tween 20 was added to the fixing solution and PBS cleaning solution to reduce the surface tension of the liquid, and prevent the dirt from depositing on the body surface of the woodboring beetle, which made the body surface cleaner in SEM.

Using TEM, sensilla on different organs of insects can be sliced to reveal the clear structures inside them, thus providing a basis for analyzing sensilla functions. When the subject insect, such as woodboring beetle, is large, and its body wall has a substantial degree of sclerotization, so the fixative may not fully saturate organ tissues inside the insect body. Tween 20 can enhance the dispersion and suspension capacity of the dirt. In this study, Tween 20 was added to the fixative to enhance fixative fluid penetration into the insect body wall of woodboring beetle, avoiding deformation and collapse of the epidermi^11,12,13. In addition, using general slicing technology, it is difficult to accurately locate different types of sensilla, in particular for some small sensilla¹⁵. Based on traditional TEM sample preparation, this study combined fluorescence microscopy and SEM to determine the position of insect sensilla in the embedded block, thus improving slicing accuracy.

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 0.1 mol L^-1 phosphate-buffered saline (PBS, pH 7.2), 2.5% (wt/vol) glutaraldehyde (Anhydrous EM Grad), and 0.06% (vol/vol) Tween 20. Fix the sample at 4 °C over the weekend.
   2. Remove the bodies from the preservation liquid and rinse in phosphate buffer. Using an stereomicroscope, remove the appendages and clean them ultrasonically (40 kHz) in a 0.1 mol L^-1 phosphate-buffered saline (pH 7.2) with 0.06% (vol/vol) Tween 20 (PBST). After cleaning for 100 s, transfer the sample to the microscope to check if it was clean. Under normal circumstances, clean for 400s to ensure that the sample was clean enough to observe and not damaged.
2. Sample Dehydration, Mounting and Drying
   1. Dehydrate the samples by using 20 min successive treatments in 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, and 100% (all vol/vol) ethanol. Under a stereomicroscope, use carbon double-sided adhesive tape to separately fix 3 observation surfaces (dorsal ventral and lateral) onto stubs. Note that all viewing surfaces must be kept clean and free of contamination. Place the sample stage in a petri dish containing a silica gel desiccant for 48 h.
3. Sputter-coat and Sample Insertion
   1. Using Hitachi Koki (E-1010) ion sputtering instrument, rotate MAIN VALVE to OPEN position, remove the sample chamber cover, and put the sample into chamber. Put the POWER switch on, and READY light was on. Set Sputtering time as 45 seconds, and coating thickness as 70.875 Å. Once mechanical pump vacuum dial index dropped below 7, press DISCHARGE and start spraying platinum. At the end of experiment, turn off the power supply and take the sample out of chamber. Spray film thickness: d = KIVt ("d" is the thickness of the film in the unit of " Å "; "K" is a constant, depending on the sputtered metal and gas. For example, K of air is 0.07; "I" is the unit mA of plasma flow; "V" is the voltage applied in the unit of "KV". "t" is time in seconds.
   2. Insert the stub containing the sample onto the stage of SEM. Make sure the sample stage with the sample stub had enough height to allow a good image. Open the SEM software and select a desired operating voltage, beginning at 20 kV.

2. TEM Sample Preparation and Imaging

1. Obtain and fix the sample as in steps 1.1.1 and 1.1.2.
2. Cleaning, Secondary Fixation and Dehydration
   1. Remove adult C. caragana from the preservation liquid. Using a stereomicroscope, remove the appendages, wash the samples in PBST for 3 h, and then post-fix them in 1% (wt/vol) osmium tetroxide in PBS for 1h at 25 °C. Dehydrate the samples by using 20 min successive treatments in 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, and 100% (all vol/vol) ethanol at room temperature.
3. Resin Embedding and Polymerization
   1. Embed the samples in resin in a flat embedding moulds. The sample was at the bottom of the plate and was placed as close as possible to the edge of the recessed groove. Place the label in the blank then incubate the plate containing the sample at 60 °C for 72 h. Remove the capsule from the incubator and verify that the resin had polymerized.
4. Sample Sectioning and Staining
   1. Once ensure the sample had been solidified, place each resin block under a fluorescence microscope and photograph them under blue light. Move the microscope's fluorescent light source so it irradiated the sample from above. Enable the sensilla in the resin block to be clearly observed. Photographed and measure distances to target the sensilla (Figure 1).
   2. Refer to SEM image of the palps (Figure 2A), and cut roughly the resin block with a razor blade to close the target receptor (Figure 2B).
   3. Next, using blue-light fluorescence microscopy, photograph the roughly cut resin block, adjusting the light source from above so that the sensilla was observed clearly. Green light excited by the blue light created a favorable observation. When imaging, the objective micrometer (DIV 0.01mm) was added to the fluorescence microscope stage, and then the distance of the target was measured by ImageJ software (U.S. National Institute of Health) (Figure 2C). The image ruler was made by Adobe Photoshop CS5 (Adobe Systems, Inc., San Jose, CA, USA). Then, for ultramicrotome slicing, set the cutting distance, using 50-60 nm slice thicknesses, until the target position was reached. Use fluorescence microscopy to pinpoint the target receptor.
   4. Mount the sections on Formvar-coated, 100-mesh copper grids, double-stained with uranyl acetate and lead citrate.
      1. Firstly, add 3.75 g uranyl acetate to 50 mL of 50 % methanol. Stain grids with a filtered (0.45 µm) syringe of a saturated solution of uranyl acetate at room temperature for 10 min. Cover sections during staining to block light induced precipitates. Rinse 2x in 50 % methanol; 2x filtered degassed water.
      2. Secondly, add 0.02 g lead citrate to 10 mL of degassed distilled water in centrifuge tube. Add 0.1 mL of 10 N sodium hydroxide, seal and shake to dissolve. Stain grids with a solution of lead citrate for 8 min. Centrifuge before use. Staining must be done in a carbon dioxide free environment to prevent the formation of lead carbonate precipitates. Place drops of stain on squares of plastic petri dishes. Rinse in degassed filtered water and dry¹⁷. Observe them via TEM operating at 80 kV.

Figure 1: A fluorescent microscope photographed a resin block enclosing the appendage of the Chlorophorus caragana. (A) Antenna resin block; (B) Resin block at the end of the ovipositor. The arrow indicated the edge of the resin block; dotted circle indicates the target sensilla. Please click here to view a larger version of this figure.

Figure 2: Procedures of the precise sensilla location method. (A) The 4th sub-segment of a maxillary palp of Chlorophorus caragana, the dotted circle showed the sensilla targeted by SEM. (B) The 4th sub-segment of a maxillary palp of C. caragana viewed by fluorescence microscopy. White arrow showed the roughly cut edge of the resin block and the dotted circle showed the accurate location. (C) The marked distance from the edge of the resin block to the maxillary palp target location (28 µm in this sample). Please click here to view a larger version of this figure.

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.

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Materials

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