Fabrication and Imaging of 3D Microphantom

The phantom is fabricated using two-photon laser lithography, in which a focused laser beam is scanned within liquid resin. The resin within the laser’s focal volume is locally polymerized. Adjusting the scanning trajectory and the exposure time of the laser beam enables simultaneous control over the 3D printed geometry (accuracy at the order of 100 nm) and RI (accuracy at the order of $5\times {10}^{-4}$ , maximal $\mathrm{\Delta }$ RI = 0.03 within the structure) in three dimensions. We used Photonic Professional GT (Nanoscribe GmbH) equipped with a 1.3 NA 100 $\times$ microscope objective and piezo scanning stage. The phantom is fabricated in the IP-Dip resin (Nanoscribe GmbH) on top of a #1.5H coverslip (dip-in configuration⁵⁴ ). After fabrication the structure was developed in PGMEA (Propylene glycol monomethyl ether acetate; 12 min), followed by isopropyl alcohol (10 min) and then blow-dried. The full methodology for fabrication and validation of the features can be found in our previous work^{33 (link)}.
To conduct our TPM imaging experiments, the microphantom was immersed in Zeiss Immersol 518F oil (RI ${}_{632}$ nm = 1.5123), which provides similar RI contrast as in the case of cells immersed in culture medium. By using immersion oils with varying RI, it is possible to adjust the scattering properties of the microphantom post-fabrication.