Apr 28, 2026
Version 2
SIM imaging and analysis for active zone release sites in vulnerable dopamine axons V.2
- Chuyu Chen1,
- Oscar Andres Moreno-Ramos1,
- Rajeshwar Awatramani1,
- Loukia Parisiadou1
- 1Northwestern University, Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD 20815
Protocol Citation: Chuyu Chen, Oscar Andres Moreno-Ramos, Rajeshwar Awatramani, Loukia Parisiadou 2026. SIM imaging and analysis for active zone release sites in vulnerable dopamine axons. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1mpdpvr2/v2Version created by Chuyu Chen
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 28, 2026
Last Modified: April 28, 2026
Protocol Integer ID: 315886
Keywords: ASAPCRN, structured illumination microscopy (SIM) , vulnerable dopamine axons vulnerable dopamine neuron, dopamine axon, structured illumination microscopy, striatal brain section, dopamine neuron, vulnerable dopamine neuron, simultaneous labeling of aldh1a1, axons from the same mouse, neuronal with mcherry, prominent in vulnerable aldh1a1, vulnerable aldh1a1, dopaminergic, neuron, axon, dopamine, aldh1a1, sim imaging, brain section
Funders Acknowledgements:
Aligning Science Across Parkinson's [ASAP-020600] through the Michael J. Fox Foundation for Parkinson's Research (MJFF)
Grant ID: ASAP-020600
Abstract
Vulnerable dopamine neurons show higher LRRK2 expression, we wondered whether active zone changes may be prominent in vulnerable Aldh1a1+ as compared to resilient Aldh1a1- dopaminergic axons in Lrrk2G2019S mice. To accomplish this, we used a intersectional/subtractive approach that allows for simultaneous labeling of Aldh1a1+ with EYFP and Aldh1a1- neuronal with mCherry, respectively. Striatal brain sections were then stained with antibodies against bassoon to analyze the number and organization of active zone sites in Aldh1a1+ (EYFP+) or Aldh1a1- (mCherry+) axons from the same mouse (Figure 6B). Due to the high signal of bassoon in the striatum, we used structured illumination microscopy (SIM) for improved resolution, followed by 3D reconstruction analysis.
Safety warnings
These protocols need prior approval by the users' Institutional Animal Care and Use Committee (IACUC) or equivalent ethics committee
Stereotactic injections and viruses
Adult mice expressing Aldh1a1/Anxa1 Cre and FlpO were anesthetized with 3% isoflurane, which was maintained at approximately 1.5% during the procedure.
Pain management included subcutaneous injections of Rimadyl (3 mg/kg) at the beginning of the surgery, followed by a slow-release injection of Buprenex (0.25 mg/kg) after the surgery, and an additional dose of
Rimadyl (3 mg/kg) the following day.
A 0.5-1 mm hole was drilled into the skull at the stereotactic coordinates of the substantia nigra pars compacta (SNc) (RC: -3.16 mm, ML: -1.50 mm, DV: -4.00 mm).
A 0.5 µl Hamilton Neuros syringe left in place for 5 minutes after reaching the target depth.
A volume of 0.3 µl of the viral vector was slowly injected into the SNc over a period of 5 minutes
a 1:1 mixture of AAV8 hSyn-Con/Fon-YFP (Addgene #55650, titer: 2.4x10¹³ GC/ml) and AAV8 Ef1a-Coff/Fon-mCherry (Addgene #137134, titer: 2.2x10¹³ GC/ml) delivered unilaterally for imaging experiments
5-minute wait to allow for proper viral diffusion within the brain parenchyma.
SIM imaging and analysis
Striatal sections (30 μm ) were incubated in 5% goat serum with 0.2% Triton X-100/PBS for 2 hours.
washe with PBS for 5 minutes x2
Probed with the primary antibodies anti-EGFP (1:1000, Invitrogen), anti-mCherry (1:1000, Invitrogen), and anti-bassoon (1:300, Enzo). 48 to 72 hours at 4°C
washe with PBS for 5 minutes x2
Incubated with secondary antibodies—Alexa Fluor‱ 488, Alexa Fluor‱ 568, and Alexa Fluor‱ 647 (1:300, Invitrogen)
Multichannel SIM images were obtained with a Nikon N-SIM Structured Illumination super-resolution microscope with a 100x objective lens (numerical aperture (NA) 1.4).
Nine images captured with 2D SIM and reconstructed with 3D SIM to increase xy and z resolution were generated by Nikon Element, and the illumination modulation contrast was set automatically by the software.
Crop images using 3D crop function to remove the edges. (resolution 1950x1950 was applied for further analysis with Imaris 10.1.
Apply resolution of 1950 x 1950 with Imaris 10.1.
Use the surface creation tool for bassoon particles and axons segmentation.
Axons segmentation: the smoothing surface detail option was enabled (Surface Grain Size = 0.0643µm). Background subtraction was performed using local contrast with a diameter of "Largest Sphere" set to 0.241µm
Apply auto-threshold value with user adjustments made as required. Set the filter “Number of Voxels Img=1” above the lower automatic threshold.
Remove the fluorescence signal outside the surface: “Mask channel” function, selecting the corresponding channel for either the Alexa488 (EGFP) or Alexa568 (mCherry), set the voxel intensity outside the surface to 0.
Measure the axonal terminal segments' length with filaments tool. In the tool wizard, the source channel for axonal terminal segments was again selected corresponding to the masked Alexa488 (EGFP) or masked Alexa568 (mCherry), with the seed points threshold set at 6,000.
Perform machine learning pixel classification. Discard unwanted segments (not overlapping with surface) while keeping segments that overlapped with the surface until they covered over 99% of visible surface
Bassoon particles segmentation: smoothing surface detail was activated (Surface Grain Size = 0.06µm).
Background subtraction (local contrast) (Diameter of Largest Sphere =0.15µm) was employed
An auto-threshold value was applied with user adjustments as required. Region Growing Estimated Diameter was set to 0.100 µm based on intensity.
Filter “quality” was set to automatic thresholds, Filter “volume” was set to between 0.003-0.04 µm3.
Bassoon particle with filter "overlapped volume ratio to surface EGFP/mCherry" was set above 1.0.
The number of bassoon objects, their volume, and the length of axon terminal segments were automatically measured and calculated.
Density calculations were performed in Excel using the number of bassoon objects relative to the total length of axonal terminal segments, with bassoon object number and axonal terminal segments length determined in Excel.