High specificity and selectivity of fibrinogen micropatterning on PLL-PEG surfaces. (A) Fibrinogen-Alexa546 (50 µg/ml) and NeutrAvidin-Dylight-550 (50 µg/ml) were micropatterned on PLL-PEG–coated glass using LIMAP with identical UV exposure and micropattern shape. After washing, red fluorescence of the micropatterns was imaged by TIRF microscopy (TIRFM) using identical settings (left and middle). Alternatively, a higher dynamic range lookup table was applied to the image on the right. (B) Quantification of the effects seen in A. Mean ± SEM of the selectivity and homogeneity (see Materials and methods). Statistics were performed using a Mann–Whitney rank-sum test. n, number of micropatterns measured. Fibrinogen quantitatively micropatterns better than NeutrAvidin. (C) Scheme illustrating the different steps for sequential multiplexed micropatterning of three fibrinogens labeled with different fluorophores (ATTO488, Alexa546, and Alexa647). (D) Multiplexed micropatterning of fibrinogen-ATTO488, Alexa546, and Alexa647 (50 µg/ml) using the scheme depicted in C onto PLL-PEG–coated glass. Note that there is high specificity of the fibrinogen for their specific micropattern and minimum overlap between the three fluorescent fibrinogens. (E) Quantification of the effects seen in D. The fluorescence of each fibrinogen was measured on the three successive micropatterns and normalized to the intensity of their respective micropattern. Mean ± SEM. Statistics were performed using a one-way ANOVA test followed by a Tukey post hoc test (P < 0.0001). n, number of micropatterns measured. Note that the amount of fibrinogen deposited onto the nonintended micropatterns is minimal. Scale bars, 10 µm. n.s., not significant.