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Updated: Apr 4, 2026
Two-photon Imaging of Cellular Dynamics in the Mouse Spinal Cord
Published on: February 22, 2015
Endogenous Two-Photon Excited Fluorescence Provides Label-Free Visualization of the Inflammatory Response in the
Ortrud Uckermann1, Roberta Galli2, Rudolf Beiermeister3
1Neurosurgery, Carl Gustav Carus University Hospital, TU Dresden, Fetscherstraße 74, 01307 Dresden, Germany.
Abstract:
Activation of CNS resident microglia and invasion of external macrophages plays a central role in spinal cord injuries and diseases. Multiphoton microscopy based on intrinsic tissue properties offers the possibility of label-free imaging and has the potential to be applied in vivo. In this work, we analyzed cellular structures displaying endogenous two-photon excited fluorescence (TPEF) in the pathologic spinal cord. It was compared qualitatively and quantitatively to Iba1 and CD68 immunohistochemical staining in two models: rat spinal cord injury and mouse encephalomyelitis. The extent of tissue damage was retrieved by coherent anti-Stokes Raman scattering (CARS) and second harmonic generation imaging. The pattern of CD68-positive cells representing postinjury activated microglia/macrophages was colocalized to the TPEF signal. Iba1-positive microglia were found in areas lacking any TPEF signal. In peripheral areas of inflammation, we found similar numbers of CD68-positive microglia/macrophages and TPEF-positive structures while the number of Iba1-positive cells was significantly higher. Therefore, we conclude that multiphoton imaging of unstained spinal cord tissue enables retrieving the extent of microglia activation by acquisition of endogenous TPEF. Future application of this technique in vivo will enable monitoring inflammatory responses of the nervous system allowing new insights into degenerative and regenerative processes.
Insights
Multiphoton microscopy using endogenous two-photon excited fluorescence (TPEF) can detect activated microglia and macrophages in damaged spinal cord tissue without labels. This label-free imaging method shows promise for monitoring neuroinflammation in vivo.
Area of Science:
- Neuroscience
- Biomedical Imaging
- Immunology
Background:
- Microglia and macrophages are key players in spinal cord injuries and diseases.
- Label-free multiphoton microscopy offers potential for in vivo imaging of neural tissue.
- Understanding cellular responses in the injured spinal cord is crucial for developing treatments.
Purpose of the Study:
- To analyze endogenous two-photon excited fluorescence (TPEF) in pathologic spinal cord tissue.
- To compare TPEF imaging with standard immunohistochemical markers (Iba1, CD68) for microglia/macrophage activation.
- To assess the utility of label-free multiphoton microscopy for monitoring neuroinflammation.
Main Methods:
- Utilized multiphoton microscopy to image endogenous TPEF in rat spinal cord injury and mouse encephalomyelitis models.
- Compared TPEF signals with Iba1 and CD68 immunohistochemistry.
- Employed coherent anti-Stokes Raman scattering (CARS) and second harmonic generation (SHG) for tissue damage assessment.
Main Results:
- TPEF signals colocalized with CD68-positive cells, indicating activated microglia/macrophages.
- Iba1-positive microglia were observed in areas lacking TPEF.
- TPEF imaging effectively reflects the extent of microglia activation in spinal cord pathology.
Conclusions:
- Label-free multiphoton imaging of endogenous TPEF can assess microglia activation in the spinal cord.
- This technique holds potential for in vivo monitoring of neuroinflammatory responses.
- Future applications could provide new insights into nervous system degenerative and regenerative processes.
