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Sample Preparation Method of Scanning and Transmission Electron Microscope for the Appendages of Woodboring Beetle
Chapters
Summary February 3rd, 2020
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.
Transcript
Woodboring beetle their body surface are always hard in SEM. The Tween-20 is added into fixative and washing solution. The body surface of woodboring beetle In TEM, the Tween-20 can enhance fixative penetration into the body wall and better fix tissue and organ in the body.
The fluorescence microscope is helpful for improving slicing accuracy. The technique is helpful for washing body surface and penetrating into the body wall in the woodboring beetle. Thus, it would clear SEM and TEM vision.
Fluorescence microscope is helpful for improving slicing accuracy in TEM. Demonstrating the procedure will be Zhang Yanru, a graduate student from our laboratory. In an area where Chlorophorus caragana live, place field traps baited with plant attractants such as isophorone on a funnel trap.
Attract woodboring beetle adults into the traps. Preserve the clean bodies of adult Chlorophorus 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. Fix the sample at four degrees Celsius for three days.
Remove the bodies from the preservation liquid and rinse in PBS. Under a stereo microscope, use forceps to remove the appendages and clean them ultrasonically at 40 kilohertz in PBST. After cleaning for 100 seconds, transfer the sample to the microscope to check if it is clean.
Do not clean longer than 400 seconds to avoid damage. Then immerse the samples in ethanol at a series of concentrations for 20 minutes each to perform dehydration. After the dehydration, under a stereo microscope, 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. Place the sample stage in a Petri dish containing a silica gel desiccant for 48 hours. 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.
Turn the power switch on and ensure the ready light is on. Set the sputtering time as 45 seconds and the coating thickness as 70.875 angstrom. Once the mechanical pump vacuum dial index drops below seven, press discharge and start spraying platinum.
At the end of the procedure, turn off the power supply and take the sample out of the chamber. 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.
Insert the stub containing the sample onto the stage of the SEM. Make sure the sample stage with the sample stub has enough height to allow a good image. Open the SEM software and select the desired operating voltage beginning at 20 kilovolts.
After obtaining the appendages from adult Chlorophorus caragana, wash the samples in PBST in an ultrasonic bath for three hours and then post-fix them in 1%weight by volume osmium tetroxide in PBS for one hour at 25 degrees Celsius. 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. With forceps, apply heated resin in a flat embedding mold and embed the samples in the resin.
Ensure that the sample is at the bottom of the plate and placed as close as possible to the edge of the recessed groove. Label and then incubate the plate containing the sample at 60 degrees Celsius for 72 hours. After incubation, remove the plate from the incubator and verify that the resin has polymerized.
Once the sample has solidified, place each resin block under a fluorescence microscope, move the microscope's fluorescent light source to irradiate the sample with blue light from above and photograph them. Make sure the sensilla in the resin block are clearly visible. Measure the distance between the edge of the resin block and the target sensor to target the sensilla.
Refer to the SEM image of the palps and roughly cut the resin block with a razor blade close to the target receptor. Next, under a blue light fluorescence microscopy, adjust the light source from above so that the sensilla are observed clearly. Add the objective micrometer to the fluorescence microscope stage.
Photograph the roughly cut resin block and then using ImageJ measure the distance between the target and the edge of the resin. Place the resin sample on an ultramicrotome and cut 50 to 60 nanometer thick sections until the target position has been reached. Mount the sections on formvar-coated 100 mesh copper grids in Petri dishes.
Stain the grids with a saturated filtered solution of uranyl acetate at room temperature. Cover the sections during staining to block light-induced precipitates. Stain for 10 minutes.
In a fume hood, rinse twice in 50%methanol, then twice in filtered degassed water. Place drops of prepared lead citrate stain on the squares of the plastic Petri dishes and let them sit for eight minutes. Rinse in degassed filtered water and dry.
Mount the grids onto a sample stage within the TEM machine. Set the voltage to 80 kilovolts and examine the sections. In this protocol, using cleaning and fixative solution with Tween-20, a clear SEM image was observed than that without Tween-20.
With the Tween-20 fixing solution allowing the glutaraldehyde fixing solution to penetrate the tissue, microtubule structures were clearly seen in the TEM image while the internal structure of the sample prepared without Tween-20 was blurry. Continuous cross-sectional views of the peg of the 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. Seven unbranched outer dendritic segments existed inside the inner receptor lymph cavity which was surrounded by an outer cavity.
The tubular body was separated by a dendritic sheath from other outer dendritic segments at each sensillar socket base. In the ciliary region, eight dendrites of different diameters were identified indicating the presence of eight bipolar neurons. The technique can be used to further study the function of nerves such as single cell recording or in situ hybridization.
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