An unbiased sampling protocol is vital for electron microscopy. If you would like to quantify it on numerical density of synapses or other structures in a certain brain area, the main advantage of our technique is that it is unbiased, and enables to automatically produce electron micrographs with minimal user intervention. This technique is also valuable in other research areas, such as neuroscience, where it can be used in assessing the strength of a neural network.
In order to apply our protocol, it's necessary to have experience in electron microscopic techniques, such as sample preparation and operating a transition electron microscope. Demonstrating our procedure will be Dr.Stefan Wernitznig, a post-doc from my laboratory. This protocol consists of preparing brain samples for electron microscopy, cutting pairs of thin sections.
Next, it will describe how to automatically produce a defined number of electron micrographs in an area of interest. Then, it will use a counting frame to assess the numerical density of structural features. To begin, dissect, fix, post-fix, and embed samples of interest into embedding resin by following along with the accompanying text protocol.
Then, place the sample onto an ultramicrotome and produce 55 nanometer ultra thin serial sections. Place the sections on a slot grid with the dimensions of one millimeter by two millimeter, coated with piola form. Then, counterstain the ultra thin sections on the slot grids using 2%uranyl acetate for 30 minutes, followed by lead citrate for 30 seconds at room temperature.
Place the grid into the TEM and examine the sections on the grid with the TEM using a low magnification to orient and evaluate the quality of the sections. Then, start the TEM Serial Section software, select Section and choose Insert to add the corner points for the reference and lookup section. Follow the instructions of the pop-up window.
Start with the reference section and then continue with the lookup section. Make sure that the edges of the section are parallel to the next section, as points one and two are entered. Next visualize the reference section under low magnification to identify the region of interest.
Move the microscope stage using TEM image analysis on the reference section to multiple corner points of the ROI to create an outline of the ROI. Record the coordinates of the resulting polygon using Random Point Sampling software. For this, press Add coordinates in the dialog box of the Random Point Sampling software at each point of the polygon that is needed to outline the ROI in the section.
Next define and enter a suitable size for the sampling areas and distances between the areas into the Random Point Sampling software. Then, press Calculate Raster to generate coordinates in a systematic, uniform, random fashion for the micrograph's positions within the polygon. Store the sampling points on the reference and lookup sections, using the Random Point Sampling software and the TEM Serial Section software.
These are the coordinates of the montages which are recorded afterwards. In the Random Point Sampling software, press go to next position to move the microscope stage to the x, y-coordinates of each sampling area in the reference section. Then, select Location and choose Insert to import these coordinates in the TEM Serial Section software in the reference section.
Repeat this for all coordinates. To mirror the coordinates from the reference section onto the lookup section, select Section and choose Go to Section. Enter the number of the lookup section in the dialog window.
For the recording of the montages, switch between the reference and lookup section, as described before, and change the position on the reference section with location and go to number. Choose the next coordinate in the dialog window. For the SerialEM montages, at each sampling coordinate, go to File and choose New Montage from the drop-down menu.
Select the right number of tiles and percentage of overlap in the dialog window. For the present study, a magnification of 5000 is sufficient to recognize the synaptic features on the study. But the limited field of view of the CCD camera required making montages of two by two images.
The montages are made to SerialEM. Before recording each montage, readjust the focus or activate the auto focus option in the recording software. Choose the folder to save the montage file and start the recording of the montage by pressing Start in the montage sub-menu, on the left in SerialEM.
Start the microdissector and define the size of the counting frame and the number of segments as needed. Count the synapse density using the dissector macro. Using the multi-point tool located in the toolbar, limit those synapses that intersect with the two forbidden lines of the dissector, but do count synapses on the opposite acceptance lines.
Mark every synapse within the counting frame that is visible in the reference section, but not visible in the lookup section, with an oval selection. For measuring synapse parameters, select only the synapses with a synaptic cleft oriented in cross-section which is on the reference section. This should be within the same image frames used for the dissector.
Next, go to Plugins and then to Analyze and start the plugin ObjectJ from the drop-down menu. Open a new project from the ObjectJ drop-down dialog which will open a window that allows the user to outline and mark structures with the marker tool. First, use the marker tool to measure the length of the pre-synaptic membrane and post-synaptic density length by drawing a line along the structure.
Then, obtain the mean width of the synaptic cleft by drawing a polygon, covering both the pre-and post-synaptic membrane. To determine the number of docked vesicles, count all vesicles that have a maximum distance from the pre-synaptic membrane of one vesicle diameter or less. Then, determine the number of undocked vesicles by counting the vesicles with a maximum distance of one vesicle diameter from docked or other undocked vesicles at the same synapse.
An adapted version of the protocol allows elemental analyses at unbiasedly obtained locations within the ultra thin section. For this, in TEM imaging mode, start SerialEM and open a new project. Then, open the Navigator and check the settings for the camera in camera and script controls for view and record and start view.
To make a corner map of the ultra thin section, choose Add Points in the Navigator window. Then, set the corner points at the edges of an ultra thin section. Move the stage to a corner point by holding down the right mouse key.
When a corner of the section is reached, add a corner point with a left mouse click. Repeat the procedure to add three more corner points. Make sure that in the Navigator window, the box C for corner points is ticked for the stored points.
To start the corner montage, go to Navigator in the SerialEM menu bar and choose Montaging Grids and Setup Corner Montage from the drop-down menu. Choose Add Polygon in the Navigator window and outline the region of interest with multiple right mouse clicks. Next, choose Add Grid of Points in the Navigator drop-down menu and define a distance between the points.
Start the EFTEMSerialEM script, follow the script commands, enter the number of grid points as shown in the Navigator and enter the number of acquisition points. Then, set the illumination threshold so that the script can avoid grid bars. Follow the script commands and move the stage manually to cover the field of view by one quarter of the grid bar.
According to the displayed value, enter a threshold illumination value that is higher than the value displayed. Wait until the points for acquisition are selected and the cooking routine is finished. This takes a long time and can be done overnight.
After setting up for energy-filtered TEM elemental analysis, acquire an elemental map with element-specific settings. The unbiased sampling approach is useful for counting and analyzing synaptic features in specific brain regions. Additionally, the EFTEMSerialEM script for the SerialEM software allows one to randomly choose the points of acquisition from within a whole ultra thin section, with the aim of obtaining an energy-filtered electron micrograph at each of these points.
The script randomly selected a number of points, pre-defined by the user from those marked with a grading. By producing a test micrograph and checking its grade levels, the script checked if the chosen points were located on a visible part of the section, rejecting the points that landed on grid bars. Most of the workflow was handled by the script, with minimal interaction of the user.
However, with the sample shown here, there was too little light for the auto focus routine of SerialEM. Therefore, as soon as the script moved the stage to each point, the focus was adjusted and an elemental map was made of iron. It is important to carefully select the region of interest and to ensure the same number of sampling points falls into each area of interest.
Do not forget that many of the chemicals used for specimen preparation are toxic. So wear a lab coat and protective gloves and work under a fume cover.
