Any life science project that requires 3D ultrastructure investigation of a specific region of interest in a complex biological sample can make use of this technique. The sample preparation procedure is the same for two imaging modalities being SBF/SEM and FIB-SEM. The use of the microwave substantially speeds up the sample processing part.
Although shown here for root tips, this protocol can be used for any biological model system with minimal adjustments. Before using this workflow for correlating SBF/SEM and FIB-SEM, one should be familiar with the individual technologies. The execution of certain steps is easily demonstrated but difficult to describe in written text.
Demonstrating the procedure will be Peter Borghgraef who is a technician in our lab. To begin, cut root tips of the fixed Arabidopsis thaliana seedlings on agar plates and put two to three tips in Eppendorf tubes containing the same fixative overnight at four degrees Celsius. In the morning, with a pipette, replace fixative with 0.1 molar phosphate buffer at pH 6.8 in the tubes with the tubes on a rotating table at 100 RPM and wash for 10 minutes.
Then repeat the washing using fresh phosphate buffer five times. In a fume hood, post-fix the root tips by replacing phosphate buffer with 2%osmium tetroxide and 0.2%ruthenium red in 0.1 molar phosphate buffer at pH 6.8 with the tubes with lids open in a microwave and start the program. Then discard the solution in the tubes and wash the root tips twice with double distilled water for five minutes each.
For the third and fourth double distilled water wash, use the microwave to assist the wash. After the first 40 second double distilled water wash, take the root tip samples out of the microwave and replace the double distilled water with fresh double distilled water. Put the samples back in the microwave and continue the program.
Next, prepare TCH solution by adding 0.1 gram of thiocarbohydrazide to 10 milliliters of double distilled water in a 15 milliliter tube. Place the tube in an oven and heat at 60 degrees Celsius for one hour to dissolve. Then filter the TCH solution using a 0.22 micrometer syringe filter.
Immediately replace the solution in the sample tubes with the filtered TCH solution and continue with the protocol. Now immerse the samples in ethanol and place them into the microwave to dehydrate in graded steps of 50%70%90%and then twice of 100%ethanol treatment. After propylene oxide dehydration treatment, add 50%Spurr's resin into the tubes to start infiltration of the root tips for a minimum of two hours.
After the final incubation in 100%Spurr's resin, place the root tips in embedding molds and fill the molds with fresh 100%Spurr's resin and polymerize in an oven at 65 degrees Celsius for 36 to 48 hours. First, use a razor blade to roughly trim the polymerized sample into a thin block. With the side having exposed tissue facing down, attach the sample to the center of a metal pin with conductive epoxy resin to have the tissue touching the metal pin.
Place the sample in an oven at 65 degrees Celsius to cure the epoxy overnight. In the morning, load the metal pin into the carrier and use a razor blade to remove any excess epoxy from the sample. To further smooth the face of the sample, load the carrier with the metal pin in the holder for the ultramicrotome.
In the ultramicrotome, use a glass or diamond knife to smooth the face of the sides of the block forming a pyramid. Make sure that at least some of the tissue is already exposed on the block face. Then place the trimmed sample block in the sputter coater and adjust the settings to platinum at two to five nanometers to coat the sample with a thin layer.
Insert the carrier in the SBF-SEM microscope. Bring the diamond knife close to the sample surface and trim off the upper portion of the sample to remove the platinum layer and expose part of the tissue. Then set the accelerating voltage to 1.5 to 2 kilovolts.
Capture an image of the tissue and set up an imaging run. Using Fiji software, select file, import, image sequence and locate the image stack to load the images. After adjusting the image according to the manuscript, check the alignment by scrolling through the data set.
Use the save command under the file menu to save the aligned data set as a 3D TIFF file. Analyze the data set carefully to see if ROI is included and contains the information that is needed for the biological question. On the last image of the stack, which is the current block face in the SBF-SEM microscope, select a new ROI for FIB-SEM.
After coating with platinum again, load the sample into the FIB-SEM and use the secondary electron detector at 15 kilovolts and one nanoamp to locate the ROI identified in the SBF-SEM on the block face. Move and tilt the stage to bring the ROI on the sample into the coincidence point of the FIB and SEM beams, using the FIB beam and gas injection system. Deposit a one micrometer protective layer of platinum on the surface above the ROI.
Next, using a low milling current ranging 50 to 100 picoamps, mill fine lines into the platinum deposition for autofocusing and 3D tracking during the imaging run. Then using a high milling current of 30 nanoamps, mill a trench of 30 micrometers in front of the ROI creating the imaging surface for the SEM beam. After smoothing the image surface, start the imaging run and monitor the stability of the process during the first 50 to 100 sections.
Once the system is running smoothly, leave the room and ensure that there is as little disturbance in the room as possible. Images from the SBF-SEM provide an overview of the tissue giving insight into the spatial orientation of cells and intracellular connections. The subsequent FIB-SEM imaging on a new region adds high resolution detail of specific cells and structures.
SBF-SEM data shows different rendering of the non-isotropic voxels from the isotropic voxel FIB-SEM data. SBF-SEM presents a staircase effect on the surface while the FIB-SEM data having the five nanometer sections ensures that the rendering appears much smoother and individual sections blend into the surface completely. This protocol describes the imaging of a unique region in one single sample and any deviation from the workflow may cause damage to the sample making it impossible to relocate the region of interest.
