These steps are covered in greater detail in the publication : A method for the three-dimensional reconstruction of Neurobiotin™-filled neurons and the location of their synaptic inputs – available online here.

STEP 1: Create a surface over the neuron

Create a surface over the neuron which best matches the neurons anatomy. To ensure all details are preserved background subtraction should be used with averaging/smoothing disabled.

1. Click create surface
2. Choose the marker delineating the neuron as the source channel. If there is no specific marker for the whole neuron you can try to use one of the markers with cytoplasmic labelling – however, be aware this may not give an accurate representation of the cell shape
3. Disable smoothing
4. Enable background subtraction
5. To determine the diameter setting for background subtraction change from surpass mode to slice mode
6. Click twice to use the line tool to measure the diameter of a dendrite on the neuron – copy this value into the background subtraction box in surpass mode
7. Adjust the surface creation threshold so that you include as much of the neuron as possible without including the background (in this case 25.8 out of 255) — Do not worry if you detect other small objects around the neuron as they can be filtered out based on size in the next step
8. Remove any small artifact surfaces which do not represent the neuron by raising the minimum voxel threshold. To do this, drag the left side of the yellow region on the graph to the right
9. Finish surface creation

STEP 2: Filter the Pre-Synaptic and Post-Synaptic Channels

Filter the pre-synaptic and post-synaptic channels so that only the fluorescent signal relevant to the filled neuron of interest remains. This requires removal of all pre-synaptic fluorescent outside the neuron and all post-synpaticfluorescence inside the neuron.

1. Select the newly created surface layer
2. Under the edit tab (pencil icon) select the button Mask All…
3. Select the pre-synaptic marker in the channel selection box
4. Enable “Duplicate channel before applying mask” to ensure the original channel data remains
5. Select constant inside/outside and check on Set voxels OUTSIDE surface to 0.0 
6. Repeat the above steps (1-5) for the post-synaptic marker, this time choosing set voxels INSIDE surface to 0.0

STEP 3: Create spot layers from the neuron filtered channels in Step 2

1. Create a new Spots layer
2. Choose the masked pre-synpatic marker channel
3. Ensure background subtraction is enabled
4. Determine the smallest diameter of the spot you wish to detect. As in the surface creation, the diameter can be easily measured by switching to Slice View and measuring with the line tool. —- here we have chosen a spot diameter of 0.3µm
5. Adjust the yellow selection area on the histogram to filter the detected spots – accurately detect as many spots as possible without creating artifacts. Generally the point of inflection on the graph yields the best results – here we have chosen 13.5
6. **if you encounter any issues with spot artifacts as a result of masking the channel with the neuron surface you can alleviate this by adding an addition filter to the detection – click + add and choose intensity mean channel X (X: the channel you are using to detect the spots)
7. Repeat steps 1-6 for the post-synaptic channel – here we used a size 0.5µm and a 14.9 for quality

STEP 4: Analysis of Pre and Post Synaptic localisations

Once both neuron specific pre and post synaptic spots have been detected two possible analyses become available:

1. Total number of pre/post synaptic spots adjacent (e.g. within 1µm) to the neuronal plasma membrane
2. Analysis of pre/post synaptic spots adjacent to each other – provides a measure of the number of functional sites

Measuring the total number of post synaptic sites proximal to the neuron:

1. Select the neuron surface
2. Choose the tools tab (red cog)
3. Select “Find spots close to surface”
4. MatLab will open and you will be prompted to choose the spots you wish to analyse – chose the spot layer which corresponds to the post-synaptic channel
5. Choose a distance threshold for the analysis – this is a distance in µms – here we have chosen 1µm
6. Two new spot layers will be created in Imaris, one with all the spots within 1µm of the neuron, the other containing all the more distant spots — in this case we detected 356 spots within 1µm of the neuron

Measuring the number of pre/post synaptic spots adjacent to each other:

1. Select either the pre or post-synaptic spot layer
2. Choose the tools tab (red cog)
3. Select “Colocalize Spots”
4. Once MatLab has opened select both the pre and post-synaptic spot layers in the menu
5. Choose a distance threshold for the analysis – this is a distance in microns between the spots – here we have chosen 1µm
6. Two new spot layers will be created in Imaris containing the pre and post-synaptic spots found within 1µm of each other — in this case we detected 45 pre-synaptic spots and 51 post-synaptic spots in a total of 32 clusters.