## 3 Results
### Integration of 3D BSA Microcomponent  
Taking advantage of 50% _w/w_ glycerol-water solutions containing BSA and MBS, the fabrication ability at the bottom of the microchannel is feasible to create a microstructure spanning the microchannel and thereby to integrate 3D microcomponents into the glass microfluidic device. In preliminary trials, we succeeded in spanning a 3D network over a 20-\(\upmu\)m-high microchannel as well as integrating a four-layered 3D structure on the ceiling of a channel by using the 200-kHz, 522-nm fiber-laser. Switching lasers to the 54-MHz oscillator laser, we were able to fabricate a four-layer woodpile structure at the ceiling of a deep microchannel (Figure 5).  
Figure 4: Writing of BSA line pattern inside a glass microchannel when using 50% _w/w_ glycerol as a solvent and a 54-MHz, 525-nm femtosecond laser. (**a**) Line width \(w\) vs. total accumulated fluence for line patterns on top of the glass surface and inside the glass channel. Solid and dashed lines indicate fitting based on a two-photon polymerization scheme with R\({}^{2}\) = 0.524 (surface) and R\({}^{2}\) = 0.741 (channel), respectively. These line widths were observed for laser powers of 3.385–6.093 mW with scanning velocities of 0.05–0.5 \(\upmu\)m/s (\(n\) = 4 for on-surface, \(n\) = 3 for in-channel fabrication). (**b**) In situ CCD camera images were modified for better visibility. Line patterns are outlined by dashed ellipses. (**i**) Inside, the channel lines written at 6.1 mW and 200 nm/s (upper) and 4.7 mW and 100 nm/s (lower) are shown. (**ii**) On the surface, two lines written at 6.1 mW and 500 nm/s are shown. Scale bars represent 10 \(\upmu\)m.  
Figure 5a illustrates a 6-\(\upmu\)m-high, four-layer woodpile design that has been employed for fabrication inside a microchannel as shown by the in situ time-lapse snapshots in Figure 5b. SEM observation after freeze-drying, metal coating, and glass cutting verified the formation of the structure at the channel ceiling (Figure 5c,d). The line pattern of one layer can be identified at all sides of the structure. Lines in the center are difficult to identify individually because the created  
Figure 5: 3D BSA woodpile structures inside glass microchannels fabricated with a 54-MHz, 525-nm laser. (**a**) Illustration of a four-layer woodpile structure with a 2-\(\upmu\)m layer pitch and a 25-\(\upmu\)m side length. (**b**) In situ CCD camera observation of the fabrication process with 6.1 mW and 100 nm/s from (**i**) one line to (**ii**) second layer, (**iii**) last layer, (**iv**) final microcomponent, and (**v**) after drying. During the drying process, the free-hanging layers were drawn closely together, causing a collapse of the expected structure. (**c**) SEM images of the view into the microchannel. The inlet, here fabricated with an offset, and channel are indicated by arrows. The position of the woodpile is highlighted by a dashed-line rectangle. Further tilting was obstructed by the inlet. (**d**) SEM view, tilted 5\({}^{\circ}\), of a woodpile structure attached inside the microchannel. (**e**–**g**) Fabrication of a 10-\(\upmu\)m woodpile: (**e**) schematic drawing, (**f**) final in situ observation, (**g**) observation after drying. The structure was lost for SEM observation when cutting the channel. Scale bars represent (**b,c**) 25 \(\upmu\)m and (**d**–**g**) 10 \(\upmu\)m.  
structure collapsed and stacked together during the drying process. Figure 5e-g illustrates how a more compact woodpile design allowed for less collapse after drying. During cutting, the channel ceiling with the structure attached broke apart. The conservation step of drying proteinaceous microstructures is already challenging for fabrication on top of the cover glass due to the attachment and low mechanical strength; moreover, drying inside the microchannel requires refined handling. Opening the microchannel for SEM observation is also difficult, because the structures need to be close to the cross-sectional cut without breaking apart.