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Optical tweezers are ideal tools for cell manipulation in vivo due to their non-contact trapping ability. In the current studies, the optical trapping of cells can only be achieved in light-permeable organs. It is important to expand the application of optical tweezers to opaque tissues and organs, where biological activities are the primary focus of biomedical research. However, the optical manipulation of cells in these opaque organs cannot be imaged using a conventional transillumination microscope due to the opacity of these organs. Here, we use optical tweezers to trap erythrocytes and measure the cell deformability in mouse liver, which are imaged with oblique back-illumination microscopy (OBM). In the microscope system, two fibers are fixed at the same oblique angle on both sides of the microscopic objective, illuminating the trapping target asymmetrically. The dual-wavelength OBM strategy allows for obtaining a differential phase contrast image in a single-shot by color channel separation. The OBM can image the flowing and trapped erythrocytes in vivo in real-time. Furthermore, the erythrocyte deformability in vivo is evaluated with optical tweezers. The spring stiffness in vivo is 8.2 ± 2.1 μN m−1, which is obtained by stretching the trapped cells with blood drag flow. Our works make it possible to utilize optical tweezers to study the live cell dynamics in opaque organs, which will expand the application of optical tweezers in the fields of biomechanical studies. [ABSTRACT FROM AUTHOR]