Microglial morphology determined with confocal and two-photon laser scanning microscopy.

Area of Science:

• Neuroscience
• Immunology
• Cell Biology

Background:

• Microglia are the primary immune cells of the central nervous system.
• Microglia morphology is a key indicator of their immune status, distinguishing between active and surveilling states.
• Accurate assessment of microglia morphology is crucial for understanding neuroinflammation and neurological diseases.

Purpose of the Study:

• To compare microglia morphology using confocal laser scanning microscopy (cLSM) and two-photon laser scanning microscopy (2P-LSM).
• To analyze differences in microglia branching patterns between in vivo and fixed tissue imaging.
• To evaluate the impact of imaging methodology on the interpretation of microglia activation states.

Main Methods:

• Investigated microglia morphology in 14-16 weeks old male transgenic mice (n=6).
• Utilized both in vivo and fixed tissue imaging with cLSM and 2P-LSM.
• Performed manual tracking and quantitative analysis of microglia branching parameters, including branch number and length across different orders.

Main Results:

• Overall microglia branching morphology showed no significant difference between cLSM and 2P-LSM.
• A higher number of fifth-order branches were observed with both cLSM and 2P-LSM in both in vivo and fixed samples.
• While largely comparable, subtle differences between imaging methods warrant consideration.

Conclusions:

• cLSM and 2P-LSM provide largely comparable data for microglia morphology analysis.
• Minor discrepancies in branch analysis between methods necessitate caution when classifying microglia as active or surveilling based solely on morphology.
• The dynamic nature of microglia requires careful consideration of the imaging technique used when labeling their activation state.