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Updated: Jan 30, 2026
Specific Labeling of Mitochondrial Nucleoids for Time-lapse Structured Illumination Microscopy
Published on: June 4, 2020
The phagocytic state of brain myeloid cells after ischemia revealed by superresolution structured illumination
Stefano Fumagalli1, Fabio Fiordaliso2, Carlo Perego1
1Department of Neuroscience, Istituto di Ricerche Farmacologiche Mario Negri IRCCS, via G. La Masa 19, 20156, Milan, Italy.
Background:
Phagocytosis is a key function of myeloid cells and is highly involved in brain ischemic injury. It has been scarcely studied in vivo, thus preventing a deep knowledge of the processes occurring in the ischemic environment. Structured illumination microscopy (SIM) is a superresolution technique which helps study phagocytosis, a process involving the recruitment of vesicles sized below the resolution limits of standard confocal microscopy.
Methods:
Mice underwent permanent occlusion of the middle cerebral artery and were sacrificed at 48 h or 7 days after insult. Immunofluorescence for CD11b, myeloid cell membrane marker, and CD68, lysosomal marker was done in the ischemic area. Images were acquired using a SIM system and verified with SIM check. Lysosomal distribution was measured in the ischemic area by the gray level co-occurrence matrix (GLCM). SIM dataset was compared with transmission electron microscopy images of macrophages in the ischemic tissue at the same time points. Cultured microglia were stimulated with LPS to uptake 100 nm fluorescent beads and imaged by time-lapse SIM. GLCM was used to analyze bead distribution over the cytoplasm.
Results:
SIM images reached a resolution of 130 nm and passed the quality control diagnose, ruling out possible artifacts. After ischemia, GLCM applied to the CD68 images showed that myeloid cells at 48 h had higher angular second moment (ASM), inverse difference moment (IDM), and lower entropy than myeloid cells at 7 days indicating higher lysosomal clustering at 48 h. At this time point, lysosomal clustering was proximal (< 700 nm) to the cell membrane indicating active target internalization, while at 7 days, it was perinuclear, consistent with final stages of phagocytosis or autophagy. Electron microscopy images indicated a similar pattern of lysosomal distribution thus validating the SIM dataset. GLCM on time-lapse SIM from phagocytic microglia cultures revealed a temporal decrease in ASM and IDM and increase in entropy, as beads were uptaken, indicating that GLCM informs on the progression of phagocytosis.
Conclusions:
GLCM analysis on SIM dataset quantitatively described different phases of macrophage phagocytic behavior revealing the dynamics of lysosomal movements in the ischemic brain indicating initial active internalization vs. final digestion/autophagy.
Insights
This study uses superresolution microscopy and image analysis to reveal distinct phases of myeloid cell phagocytosis in brain injury, differentiating active internalization from later digestion stages. This advances understanding of myeloid cell function in ischemic environments.
Area of Science:
- Neuroscience
- Cell Biology
- Biomedical Imaging
Background:
- Phagocytosis by myeloid cells is crucial in brain ischemic injury but poorly understood in vivo.
- Structured illumination microscopy (SIM) offers superresolution to study phagocytosis, including sub-resolution vesicle dynamics.
Purpose of the Study:
- To investigate myeloid cell phagocytosis dynamics in vivo during brain ischemic injury using SIM.
- To quantitatively analyze lysosomal distribution and its correlation with phagocytic activity.
Main Methods:
- Mice with induced ischemic stroke underwent imaging at 48 hours and 7 days post-insult.
- Immunofluorescence and SIM were used to visualize myeloid cells (CD11b) and lysosomes (CD68).
- Gray level co-occurrence matrix (GLCM) analyzed lysosomal distribution, validated by transmission electron microscopy and in vitro microglia cultures.
Main Results:
- SIM achieved 130 nm resolution, confirming image quality.
- GLCM analysis revealed higher lysosomal clustering near the cell membrane at 48 hours post-ischemia, indicating active uptake.
- At 7 days, lysosomal distribution shifted perinuclearly, suggesting later digestion or autophagy stages.
Conclusions:
- GLCM analysis of SIM data quantitatively differentiates early active phagocytosis from later stages in ischemic brain myeloid cells.
- The study elucidates the dynamic changes in lysosomal positioning, reflecting phagocytic progression and cellular fate.
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