Two‐Photon 3D Printing of Functional Microstructures Inside Living Cells

3D printing is transforming manufacturing and biomedicine, yet it has not been demonstrated inside living cells. Additionally, there is no method to deliver micron-scale, free-standing solid microstructures directly into the cytosol of non-phagocytic cells. Here, both of these challenges are addressed by fabricating custom-shaped polymeric microstructures directly inside living cells using two-photon polymerization. A bio-compatible photoresist is injected into cells and selectively polymerized with a femtosecond laser, creating intracellular structures with submicron resolution. Structures of various shapes are printed in live cells, including a 10 <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"> <mml:semantics><mml:mrow><mml:mi>μ</mml:mi> <mml:mi>m</mml:mi></mml:mrow> <mml:annotation>$\umu {\rm m}$</mml:annotation></mml:semantics> </mml:math> elephant, barcodes for cell tracking, diffraction gratings for remote readout, and microlasers. The printed structures in cells can affect the cell biology. The demonstrated top-down intracellular biofabrication approach, combined with functional photoresists, may enable new applications in intracellular sensing, biomechanical manipulation, bioelectronics, and targeted drug delivery. These embedded structures could provide novel control over the intracellular environment, allowing engineering of cellular properties beyond natural limits and genetic engineering.