Femtosecond laser 3D printing in three colors of fluorescent protein

We report the ability of three-dimensional (3D) printing from pure fluorescent protein in the colors red, blue and green via femtosecond laser direct write (fs-LDW) technology. We utilize the protein variants mScarlet, enhanced blue fluorescent protein (EBFP2) and enhanced green fluorescent protein (EGFP) for red, blue and green colors, respectively. The family of fluorescent protein is a well-established biological tool used for fluorescence labelling and pH indication. We demonstrate that the feature of fluorescence is retained after fabrication. We further provide details regarding the fluorescence retention, in particular which fabrication parameters achieve the best outcomes. Fs-LDW is a promising technique for fine 3D printing of biomaterials such as protein due to nonlinear multiphoton absorption processes facilitating localized microfabrication along a designated laser light path. The use of protein as a precursor material for fs-LDW is attractive because the fabricated structures retain their native functions as demonstrated by several reports. These reports range over various combination of protein and photoactivators, but pure protein can also be utilized as a precursor. The resulting proteinaceous microstructures with native function retained and submicron feature sizes might offer diverse biomedical or biochip applications. This work highlights the capabilities of fs-LDW from pure protein with a biologically highly regarded protein, broadening the scope of the 3D printing technology as well as providing a new use for the fluorescent protein family.