Two-photon 3D printing of functional microstructures inside living cells

3D printing has revolutionized numerous scientific fields and industries, with printing in biological systems emerging as a rapidly advancing area of research. However, its application to the subcellular level remains largely unexplored. Here, we demonstrate for the first time the fabrication of custom-shaped polymeric microstructures directly inside living cells using two-photon polymerization. A biocompatible photoresist is injected into live cells and selectively polymerized with a femtosecond laser. The unpolymerized photoresist is dissolved naturally within the cytoplasm, leaving behind stable intracellular structures with submicron resolution within live cells. We printed various shapes, including a $10 μm$ elephant, barcodes for cell tracking, diffraction gratings for remote readout, and microlasers. Our top-down intracellular biofabrication approach, combined with existing functional photoresists, could open new avenues for various applications, including intracellular sensing, biomechanical manipulation, bioelectronics, and targeted intracellular drug delivery. Moreover, these embedded structures could offer unprecedented control over the intracellular environment, enabling the engineering of cellular properties beyond those found in nature.