## 2 Materials and Methods
We observed line fabrication for \(n\) = 3-4 individual samples per fabrication scenario and measured line width for the laser powers of 0.0935-6.4 mW with scanning velocities of 0.2-10 um/s in the case of the 200-kHz, 522-nm laser and laser powers of 3.385-6.093 mW with scanning velocities of 0.05-0.5 um/s in the case of the 54-MHz, 525-nm laser (see Supplementary Materials for further details, Table S2). Depending on the precision of the alignment of the incidental angle to the crystal for second harmonic generation, the exact maximum laser power available could vary day by day. When fabricating with high values of fluence, the minimum velocity was limited to 0.05 um/s of the mechanical stage. Exploring fabrication with lower values of fluence, scanning velocities outside the abovementioned ranges did not yield line fabrication. After fabrication, samples were submerged in water to properly rinse the microchannels. For subsequent second protein fabrication, the microchannels were gently dried in air and refilled with another working solution. For the conservation of 3D microcomponents, the glass microfluidics integrated with proteins were transferred to ethanol (special grade, 057-00456, Wako Pure Chemical Industries, Ltd., Osaka, Japan) and isopropanol (166-04836, Wako Pure Chemical Industries, Ltd., Osaka, Japan) baths, respectively, and then dried in a freeze dryer (FDS-1000, Eyela, TOKYO RIKAKIKAI CO., LTD., Tokyo, Japan). Fabricated structures were observed by optical microscope Olympus BX51 (Olympus, Tokyo, Japan) and 3D optical profiler Zeta-20 (Zeta Instruments, San Jose, CA, USA). For scanning electron microscope (SEM) observation by Jeol6330 (Jeol Ltd., Tokyo, Japan), samples were metal-coated with a palladium-platinum target (MSP-20-UM, Vacuum Device, Mito, Japan). The 1-mm-thick samples were mechanically cut at the position of the inlets perpendicularly to the microchannel direction so as not to induce damage to the protein structures formed on the about 100-um-thin microchannel glass ceiling, which allows cuts to reveal the cross-sectional view. Thereafter, samples were metal-coated again normal to the cutting cross-section to be able to image the cross-sectional view of the microchannel from the cut section. We evaluated structures and line patterns made on top of the channel surface as well as inside the channel at the ceiling. As a feature size, line width is measured from in situ images and fit against the product of the total accumulated fluence (TAF) and fluence, calculated from laser power, scanning velocity, and laser parameters. The function for fitting reflects a two-photon absorption that induces the radical generation. The dependency of the product of TAF and fluence is determined in the supplementary information using Equations (1)-(14), where the effective number of pulses [23] and line width dependence of laser power and processing time [24] are considered. The fitting results are shown in Figure S1. Since a product of TAF and fluence is not intuitive and only utilized to satisfy the equation, the fitting parameters are applied together with fluence to depict the dependency of TAF in the results.