Abstract
<title>Abstract</title> <p>Differential phase contrast (DPC) microscopy is widely used for label-free structural imaging of transparent specimens, yet whether phase-gradient signals can provide endogenous functional contrast remains unknown. Here we report dynamic reflection differential phase contrast (D-RDPC), a label-free imaging modality that reveals intracellular activity through temporal fluctuations of directional phase-gradient contrast. Using living Müller glial cells cultured on highly reflective substrates, we show that asymmetric illumination generates a directional dynamic signal that exhibits three characteristic signatures of phase-gradient imaging: contrast reversal upon illumination inversion, systematic enhancement with increasing illumination asymmetry, and recovery of spatial localization through directional Hilbert-transform reconstruction. These observations support the interpretation that the measured contrast originates from temporal fluctuations of a reflection differential phase contrast signal rather than from conventional dynamic interferometric scattering alone. D-RDPC reveals intracellular activity that remains poorly visible with conventional dynamic full-field optical coherence tomography and enables functional imaging at reflective biointerfaces where strong specular reflections typically limit performance. More broadly, our results establish temporal fluctuation analysis as a route to extending DPC microscopy from a structural imaging modality toward functional imaging.</p>