## 2 Materials and Methods  
After glycerol (G9012 Sigma-Aldrich, Tokyo, Japan) was weighed to 500 mg and deionized, purified water (Purelab, ELGA, High Wycombe, United Kingdom) was added to a total weight of 1 g to achieve a 50% _w/w_ glycerol-water mixture. BSA powder (A7906, Sigma-Aldrich, Tokyo, Japan) was weighed and appropriately diluted with water or glycerol-water to 200 mg/mL, which equates \(\sim\)3 mM. Photoinitiator MBS [18], provided by the University of Maryland, was dissolved in water at 200 mM and then mixed with BSA solution to a concentration of 100 mM MBS and 1.5 mM BSA. In order to achieve a 50% _w/w_ glycerol-water 67 mM MBS, 1.5 mM BSA mixture, several steps were performed. First, 50 mg/mL BSA was dissolved in 75% _w/w_ glycerol-water, then by mixing it with 200 mM MBS dissolved in water with a ratio of 2:1 we reached a 67 mM MBS 50% glycerol-water mixture of BSA. In order to accurately meet the 100 mg/mL BSA value, i.e., 1.5 mM BSA, for this mixture, a small amount of BSA was weighed and the 67 mM MBS-containing 50% glycerol-water mixture was pipetted and properly mixed. Although it might be possible to first mix MBS with glycerol-water and then add it to BSA powder of appropriate amounts, we performed several steps because pipetting glycerol at small volumes or weighing small MBS amounts were both inferior in accuracy to working with existing stock solutions prepared between 0.5 and 2 mL. By recombinant expression, EGFP was expressed with polyhistidine tag at N terminus from _E. coli_ JM109 (DE3) [22]. The protein was diluted with buffer solution containing 150 mM KCl and 50 mM HEPES-KOH with pH = 7.4. Concentration of EGFP was measured to be 4.7 mg/mL by a microvolume spectrophotometer BioPhotometer (Eppendorf AG, Hamburg, Germany). This solution was mixed with 20 mM MBS to obtain a final solution of 2.35 mg/mL EGFP and 10 mM MBS. In order to contrast BSA against EGFP, 20 mM of red fluorescent molecule rhodamine 6G (R6G, Junsei Chemical Co., Ltd., 87185-1610, Tokyo, Japan) was used as a photosensitizer to achieve 1 mM R6G, 200 mg/mL BSA, 50% _w/w_ glycerol-water solution. Glass microchannels were fabricated as described elsewhere [4; 5]. In brief, focused \(\upmu\)J-femtosecond laser light of either fundamental wavelength at 1045 nm or second generated harmonic wavelength at 522 nm was scanned in photo-sensitive Foturan(r) glass to 3D space-selectively modify the glass where the cavity of the microchannel should be formed. Thereafter, glass was annealed thermally in a tubular furnace (TP20-K, Thermo RIKO Co., Ltd., Tokyo, Japan) up to 605 \({}^{\circ}\)C and subsequently treated with about 8% hydrofluoric acid (HF, 082-03525, Wako Pure Chemical Industries, Ltd., Osaka, Japan) for the selective wet etching of the modified regions. Lastly, surfaces of fabricated microchannels were smoothened with a second thermal annealing in the furnace up to 620 \({}^{\circ}\)C.  
After positioning the fabricated glass microfluidics underneath the 20\(\times\) objective lens (CF Plan, Nikon Corporation, Tokyo, Japan) with numerical aperture N.A. = 0.46 and a working distance of 3.1 mm, a 5-10 \(\upmu\)L droplet of working solution was cast on the inlets. After confirming the intrusion of solution, fabrication was performed within 30 min to avoid drying and clogging of the water-dissolved samples in the microchannel. Glycerol-water-dissolved samples could be utilized within a day. We employed two femtosecond lasers in series, FCPA uJewel D-400 (IMRA America Inc., Ann Arbor, MI, USA) with 200-kHz frequency and Mikan (Amplitude Systemes, Pessac, France) with 54-MHz frequency (see Supplementary Materials for further details, Table S1) for fabrication. From the fundamental laser light, we generated a second harmonic wavelength of either 522 nm for the FCPA uJewel D-400 or about 525 nm for Mikan using a beta barium borate crystal.