Simultaneous imaging of various facets of intact biological systems across multiple spatiotemporal scales would be an invaluable tool in biomedicine. However, conventional imaging modalities have stark tradeoffs precluding the fulfilment of all functional requirements.Here we propose the refractive index (RI), an intrinsic quantity governing light-matter interaction, as a means for such measurement. We show that major endogenous subcellular structures, which are conventionally accessed via exogenous fluorescence labeling, are encoded in 3D RI tomograms. We decode this information in a data-driven manner, thereby achieving multiplexed microtomography. This approach inherits the advantages of both highspecificity fluorescence imaging and label-free RI imaging. The performance, reliability, and scalability of this technology have been extensively characterized, and its application within single-cell profiling at unprecedented scales has been demonstrated..

Deconvolution phase microscopy enables high-contrast visualization of transparent samples through reconstructions of their transmitted phases or refractive indexes. Herein, we propose a method to extend 2D deconvolution phase microscopy to thick 3D samples. The refractive index distribution of a sample can be obtained at a specific axial plane by measuring only four intensity images obtained under optimized illumination patterns. Also, the optical phase delay of a sample can be measured using different illumination patterns.

Phosphate-buffered saline (PBS) and Alsever’s solution (AS) are frequently used as media in blood-related studies, while 0.9% normal saline (NS) is frequently used in transfusion medicine. Despite the frequent use, the effects of these solutions on the shape and volume of red blood cells (RBCs) have not been reported. We collected blood samples from five healthy adults and used three-dimensional refractive index tomography to investigate the changes in the morphology of RBCs caused by changes in osmolality and solutes at the single-cell level. After diluting 2 μL of RBCs 200-fold with each solution (PBS, AS, and 0.9% NS), 40 randomly selected RBCs were microscopically observed. RBC shape was measured considering sphericity, which is a dimensionless quantity ranging from 0 (flat) to 1 (spherical). RBCs in plasma or AS showed a biconcave shape with a small sphericity, whereas those in 0.9% NS or PBS showed a spherical shape with a large sphericity. Moreover, we confirmed that sodium chloride alone could not elicit the biconcave shape of RBCs, which could be maintained only in the presence of an osmotic pressure-maintaining substance, such as glucose or mannitol. Although 0.9% NS solution is one of the most commonly used fluids in hematology and transfusion medicine, RBCs in 0.9% NS or PBS are not biconcave. Therefore, as the debate on the use of NS continues, future clinical studies or applications should consider the effect of glucose or mannitol on the shape of RBCs.

We report a deep-learning-based imaging technique to predict 3D multiplexed fluorescence signals based on label-free refractive index measurements. We demonstrate the retrieval of specific subcellular information without exogenous labeling or staining, enabling multiplexed molecular imaging across various spatiotemporal scales.