Protocol for <i>in vivo</i> fluorescence lifetime microendoscopic imaging of the murine femoral marrow

Abstract We present a protocol for micro-endoscopic fluorescence lifetime imaging in the femoral marrow of mice allowing the analysis of NAD(P)H-dependent metabolism at single cell level, in vivo . Therefore, we employed a gradient refractive index (GRIN) lens system fixed to the mouse femur by a specialized implant. We provide step-by-step instructions for the practical usage of the microendoscopic femoral implant and discuss experimental parameters required for reliable NAD(P)H FLIM analysis, particularly referring to photon statistics and signal-to-noise ratio. Representative results indicate metabolic heterogeneity both in marrow tissue environment and among marrow LysM + myeloid cells in vivo . We expect the here presented microendoscopic FLIM approach to enable the analysis of cellular functions and dysfunctions, beyond cellular metabolism, providing a better understanding of bone biology, in health and disease.