Submission ID #: 59251

Scriptwriter Name: Qingyun Ping

Project Page Link: https://www.jove.com/account/file-uploader?src=18071603

Title: Sample Preparation of Woodboring Beetle Appendages for Electron Microscopy

Authors and Affiliations: Yan-Ru Zhang1,2, Hai-Li Qiao3, Li-Li Ren1, Rong Wang4, Peng-Fei Lu1

1The Key Laboratory for Silviculture and Conservation of the Ministry of Education, School of Forestry, Beijing Forestry University, Beijing, People’s Republic of China

2School of Forestry, Inner Mongolia Agricultural University, Hohhot, People’s Republic of China

3Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, People’s Republic of China

4School of Forestry, Fujian Agriculture and Forestry University, Fuzhou, People’s Republic of China

Corresponding Author:

Peng-Fei Lu

lpengfei224@126.com

Email addresses for Co-authors:

zhangyanru4479@126.com

qhl193314@sina.com

lily_ren@bjfu.edu.cn

442828653@qq.com

Author Questionnaire:

1. Microscopy: Does your protocol involve video microscopy, such as filming a complex dissection or microinjection technique? (Y/N)

N

Can you record movies/images using your own microscope camera? (Y/N)

Y

2. Does your protocol include software usage? (Y/N)

If yes, we will need you to record using screen recording software to capture the steps. If you use a Mac, QuickTime X also has the ability to record the steps.

N

3. Which steps from the protocol section below are the most important for viewers to see? Please list 4-6 individual steps using the step numbers listed in this document. This information is important to prepare your Videographer for your shoot. (You do not need to include steps that will be screen captured. Please do not list entire sections.)

2.3 2.4 3.3 3.5 3.6 3.7

4. What is the single most difficult aspect of this procedure and what do you do to ensure success? Please list 1-2 individual steps using the step numbers listed in this document. (Please do not list entire sections.)

3.5

5. Will the filming need to take place in multiple locations? (Y/N)

If yes, how far apart are the locations?

Y 10KM

Section - Introduction

Videographer: Interviewee Headshots are required. Take a headshot for each interviewee.

Authors, these headshots will be used for the JoVE Dedicated Author Webpage. Here is one example if you wish to take a look.

REQUIRED Interview Statements: (Said by you on camera) - All interview statements may be edited for length and clarity.

Pengfei Lu: woodboring beetle are large, their body surface often have various pollutants, and their body wall have a substantial degree of sclerotization. In SEM, Tween 20 is added into the fixative and the wash solution, the insect body surface of woodboring beetle is washed more cleanly. In TEM, Tween 20 can enhance fixative fluid penetration into the insect body wall and subsequently better fix tissues and organs in the body. Fluorescence microscopy is helpful for improving slicing accuracy [1].

INTERVIEW: Named author says the statement above in an interview-style shot while looking slightly off-camera.

Pengfei Lu: This technique is helpful for washing body surface and penetrating into the insect body wall of woodboring beetle, thus yield clear SEM and TEM observations. Fluorescence microscopy is helpful for improving slicing accuracy in TEM [1].

INTERVIEW: Named author says the statement above in an interview-style shot while looking slightly off-camera.

Introduction of Demonstrator: (Said by you on camera)

Dr. Pengfei Lu: Demonstrating the procedure will be Dr. Yanru Zhang, a grad student from my laboratory [1] [2].

Interview style: Author saying the above

The named technician, post doc, student looks up from workbench or desk or microscope and acknowledges the camera.

Section - Protocol

SEM Sample Preparation and Imaging

In an area where C. caragana (pronounce as Chlorophorus caragana) live, place field traps baited with plant attractants, such as isophorone, on a Funnel trap [1]. Attract Woodboring Beetle adults into the traps [2].

WIDE: Talent places plant attractant on funnel traps.

LAB MEDIA: Shot of beetles in the trap. 2.1.2_2.jpg

Preserve the clean bodies of adult C. caragana in 0.1-mole per liter PBS at pH 7.2, 2.5% weight by volume glutaraldehyde, and 0.06% volume by volume Tween 20 (pronounce as tween twenty) [1]. Fix the sample at 4 degrees Celsius for 3 days [2].

CU: Talent transfers the beetles into a tube with solution.

Talent places the tube into a refrigerator.

Remove the bodies from the preservation liquid and rinse in the PBS [1]. Under a stereomicroscope, use forceps to remove the appendages [2] and clean them ultrasonically at 40 kilohertz in PBST (pronounce as P-B-S-T) [3].

CU: Talent transfers the beetles from the tube to another tube.

SCOPE: Talent removes the appendages. Important Step

Talent places the appendages in ultrasonic bath. Important Step

After cleaning for 100 seconds, transfer the sample to the microscope to check if it is clean [1]. Do not clean longer than 400 seconds to avoid damage [2].

Talent places the sample under a microscope to check. Important Step

SCOPE: Shot of the cleaned sample.

Then, immerse the samples in ethanol at a series of concentrations for 20 minutes each to perform dehydration [1-TXT].

Talent places the sample into a tube with ethanol. TEXT: 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, and 100% (v/v) Videographer: Take multiple shots, as this will be used later.

After the dehydration, under a stereomicroscope, use carbon double-sided adhesive tape to separately fix three observation surfaces, dorsal, ventral, and lateral onto stubs. Ensure that all viewing surfaces are kept clean and free of contamination [1]. Place the sample stage in a Petri dish containing a silica gel desiccant for 48 hours [2].

SCOPE: Talent fixes the samples onto stubs, and shows the surfaces.

Talent places the stage in a dish with desiccant.

Next, on an ion sputtering instrument, rotate the main valve to the open position, remove the sample chamber cover, and put the sample into the chamber [1]. Turn the power switch on, and ensure the ready light is on [2].

Talent operates on the ion sputtering instrument, and puts sample into the chamber.

CU: Talent turns on power switch, and shows the ready light.

Set the sputtering time as 45 seconds, and the coating thickness as 70.875 Angstrom [1]. Once the mechanical pump vacuum dial index drops below 7, press DISCHARGE and start spraying platinum [2].

CU: Talent adjusts sputtering time and coating thickness.

Shot of the index < 7, and then talent starts the spraying.

At the end of the procedure, turn off the power supply and take the sample out of the chamber [1]. Calculate the spray film thickness d in Angstrom, equaling k times i times v times t. K is a constant, determined by the sputtered metal and gas, i is the plasma flow in milliamps, while v is the voltage applied in kilovolts, and t is the time in seconds [2].

Talent turns off power supply and takes out the sample.

Talent does calculation on a paper. d = KIVt

Insert the stub containing the sample onto the stage of the SEM [1]. Make sure the sample stage with the sample stub has enough height to allow a good image [2]. Open the SEM software and select a desired operating voltage, beginning at 20 kilovolts [3].

Talent inserts the stub onto the SEM stage.

CU: Shot from the side to show the height of the sample stage.

SCREEN: Talent opens SEM software and selects operating voltage

TEM Sample Preparation and Imaging

After obtaining the appendages from adult C. caragana [1], wash the samples in PBST in an ultrasonic bath for 3 hours [2], and then post-fix them in 1% weight by volume osmium tetroxide in PBS for 1 hour at 25 degrees Celsius [3].

CU: Talent shows the appendages. Author comment: for the clean appendages, shots 2.1-2.4 should be carried out again. But these steps were repeated and not filmed here.

CU: Talent places the samples into a tube with solution.

Talent transfers the sample from the tube to another tube.

Dehydrate the samples by using 20-minute successive treatments in 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, and 100% volume by volume ethanol at room temperature [1-TXT].

Use 2.5.1. Talent places the sample into a tube with ethanol. TEXT: 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, and 100% (v/v) Videographer: Take multiple shots, as this will be used later. Author comment: same for shot 2.5. But the step was repeated and not filmed here.

With forceps, apply heated resin in a flat embedding mold and embed the samples in the resin [1]. Ensure that the sample is at the bottom of the plate and placed as close as possible to the edge of the recessed groove [2]. Label and then incubate the plate containing the sample at 60 degrees Celsius for 72 hours [3].

CU: Talent places the samples in a mold with resin. Important Step

ECU: Shot of the sample at the bottom and close to the edge of the groove. Important Step

Talent labels the plate and places into an incubator.

After incubation, remove the plate from the incubator and verify that the resin has polymerized [1].

Talent takes out the plate and shows the resin has polymerized.

Once the sample is solidified, place each resin block under a fluorescence microscope [1], move the microscope’s fluorescent light source to irradiate the sample with blue light from above [2], and photograph them. Make sure the sensilla in the resin block are clearly visible [3].

Talent places the block under a microscope.

Talent adjusts the light source. Important Step

SCOPE: Shot of the sample under blue light. Important Step

Measure the distance between the edge of the resin block and the target sensor to target the sensilla [1-LM]. Refer to the SEM image of the palps [2-LM] and roughly cut the resin block with a razor blade close to the target receptor [3].

LAB MEDIA: Figure 1

LAB MEDIA: Figure 2A

SCOPE: Talent cuts the resin block. Important Step

Next, under a blue-light fluorescence microscopy, adjust the light source from above so that the sensilla are observed clearly [1]. Add the objective micrometer to the fluorescence microscope stage, photograph the roughly cut resin block [2], and then using ImageJ, measure the distance between the target and the edge of the resin [3].

SCOPE: Talent shows the cut resin block, with view of the sample. Important Step

Talent attaches objective micrometer. Important Step

SCREEN: Talent measures distance.

Place the resin sample on an ultramicrotome [1], and cut 50-60 nanometers thick sections, until the target position has been reached [2]. Mount the sections on Formvar-coated, 100-mesh copper grids in Petri dishes [3].

Talent places the resin on an ultramicrotome.

Talent cuts sections.

CU: Talent places the sections onto grids.

Stain the grids with a saturated filtered solution of uranyl acetate at room temperature [1-TXT]. Cover the sections during staining to block light-induced precipitates. Stain for 10 minutes [2].

CU: Talent adds solution over the grids. TEXT: See manuscript for the solution preparation.

Talent covers the sections.

In a fume hood, rinse twice in 50% methanol then twice in filtered degassed water [1]. Place drops of prepared lead citrate stain on the squares of the plastic Petri dishes, and let them sit for 8 minutes [2]. Rinse in degassed filtered water and dry [3].

Talent adds solvent onto the grids.

CU: Talent drips stain solution onto dishes.

Talent rinses the grids in water, takes them out, and places them on a surface to dry.

Mount the grids onto a sample stage within the TEM machine, set the voltage to 80 kilovolts [1], and examine the sections [2].

Talent places the grids in the TEM machine, and adjusts the voltage.

SCREEN: Talent shows the sample.

Section – Results

Results: SEM and TEM

In this protocol [1], using cleaning and fixative solution with Tween 20, a clearer SEM image [2] was observed than that without Tween 20 [3].

LAB MEDIA: Figure 3

LAB MEDIA: Figure 3 – Video editor: emphasize Figure 3A.

LAB MEDIA: Figure 3 – Video editor: emphasize Figure 3B.

With the Tween 20 fixing solution allowing the glutaraldehyde fixing solution to penetrate the tissue, microtubule structures were clearly seen in the TEM image [1]. While the internal structure of the sample prepared without Tween 20 was blurry [2].

LAB MEDIA: Figure 4 – Video editor: emphasize Figure 4A.

LAB MEDIA: Figure 4 – Video editor: emphasize Figure 4B.

Continuous cross-sectional views of the peg of the S.tb.1 (pronounce as Type one sensilla twig basiconica) on the maxillary palps showed that the dendritic sheath surrounded the outer dendritic segments and extended to the tip pore [1].

LAB MEDIA: Figure 5 - Video editor: emphasize the dotted line E in figure 5A and Figure 5E at the same time, then emphasize the dotted line D in figure 5A and Figure 5D at the same time, then the dotted line C in figure 5A and Figure 5C at the same time, then the dotted line B in figure 5A and Figure 5B at the same time.

Seven unbranched outer dendritic segments existed inside the inner receptor lymph cavity, which was surrounded by an outer cavity [1]. The tubular body was separated by a dendritic sheath from other outer dendritic segments at each sensillar socket base [2].

LAB MEDIA: Figure 5 - Video editor: emphasize the Figure 5D.

LAB MEDIA: Figure 5 - Video editor: emphasize the Figure 5E.

In the ciliary region, eight dendrites of different diameters were identified, indicating the presence of eight bipolar neurons [1].

LAB MEDIA: Figure 5 - Video editor: emphasize the Figure 5F. 4.5.1.

Section - Conclusion

Conclusion Interview Statements: (Said by you on camera) - All interview statements may be edited for length and clarity.

Pengfei LU: This technique can be used to further study the function of nerves, such as single-cell recording or in situ hybridization [1].

INTERVIEW: Named author says the statement above in an interview-style shot while looking slightly off-camera.