MarShie: a clearing protocol for 3D analysis of single cells throughout the bone marrow at subcellular resolution


  • T.F. Mertens
  • A.T. Liebheit
  • J. Ehl
  • R. Köhler
  • A. Rakhymzhan
  • A. Woehler
  • L. Katthän
  • G. Ebel
  • W. Liublin
  • A. Kasapi
  • A. Triantafyllopoulou
  • T.J. Schulz
  • R.A. Niesner
  • A.E. Hauser


  • Nature Communications


  • Nat Commun 15 (1): 1764


  • Analyzing immune cell interactions in the bone marrow is vital for understanding hematopoiesis and bone homeostasis. Three-dimensional analysis of the complete, intact bone marrow within the cortex of whole long bones remains a challenge, especially at subcellular resolution. We present a method that stabilizes the marrow and provides subcellular resolution of fluorescent signals throughout the murine femur, enabling identification and spatial characterization of hematopoietic and stromal cell subsets. By combining a pre-processing algorithm for stripe artifact removal with a machine-learning approach, we demonstrate reliable cell segmentation down to the deepest bone marrow regions. This reveals age-related changes in the marrow. It highlights the interaction between CX(3)CR1(+) cells and the vascular system in homeostasis, in contrast to other myeloid cell types, and reveals their spatial characteristics after injury. The broad applicability of this method will contribute to a better understanding of bone marrow biology.