Figure S1.
Fibrinogen micropatterning using LIMAP.(A) Optical design of the DMD-UV illuminator used in this study. Schematic optical path. A 385-nm high-power UV LED light source is collimated using an AR-coated aspheric lens, and the collimated UV beam is then directed toward a DMD chip at a 24° angle of incidence (corresponding to twice the tilting angle of the DMD mirrors). The image of the DMD chip is then relayed onto the conjugate of the sample plane at the backport of the microscope through a 4f imaging system (f1 = f2 = 125 mm UV fused silica bi-convex lenses, AR-coated). This intermediate image is then relayed onto the sample plane by a tube lens and the objective. To combine DMD-UV illumination with TIRF illumination, a 470-nm dichroic is placed after f2 within a custom backport assembly (Cairn). To offer a second illumination wavelength for 450-nm optogenetic stimulation, our design also contains a second 450-nm collimated LED at the symmetric −24° angle. This LED can be exchanged for any other LED to provide epifluorescence imaging. (B and C) Enhanced fibrinogen micropatterning efficiency depends on the buffer used. (B) PLL-PEG–coated glass was processed for LIMAP patterning with identical UV exposure, micropattern shape, and photoinitiator concentration (50 mM BBTB in 0.1 M sodium bicarbonate, pH 8.3). Fibrinogen-Alexa546 (50 µg/ml) was then adsorbed onto the UV-activated surface in either PBS or carbonate buffer. After washing, red fluorescence of the patterns was imaged by TIRFM using identical settings. (C) Quantification of the effects seen in B (average selectivity ± SEM; see Materials and methods). Fibrinogen quantitatively patterns better in carbonate buffer. Statistics were performed using a Mann–Whitney rank-sum test. n, number of patterns measured. (D–F) Advantages of fibrinogen for multiplexed micropatterning. (D) Scheme illustrating the different steps for sequential multiplexed micropatterning of two fibrinogens labeled with different fluorophores (ATTO488 and Alexa647). (E) Multiplexed micropatterning of fibrinogen-ATTO488 and Alexa647 (50 µg/ml) using the scheme depicted in D onto PLL-PEG–coated glass. Note that there is high specificity of the fibrinogen for their specific pattern and minimum overlap among the two fluorescent fibrinogens. In addition, binding of fibrinogen to unexposed PLL-PEG is minimal, down to a punctate, single molecule–like level (orange arrowheads). (F) Quantification of the effects seen in E: the fluorescence of each Fibrinogen was measured on the two patterns and normalized to the fluorescence of their intended pattern (i.e., first pattern for Alexa647 and second for ATTO488; mean ± SEM). Statistics were performed using a Kruskal–Wallis test followed by a Dunn post hoc test (P < 0.0001). n, number of patterns measured. Note that the vertical scale of the graph is split to better appreciate the minute amounts of fibrinogen deposited onto the nonintended patterns or the unpatterned PLL-PEG area. Scale bars, 10 µm. n.s., not significant.

Fibrinogen micropatterning using LIMAP. (A) Optical design of the DMD-UV illuminator used in this study. Schematic optical path. A 385-nm high-power UV LED light source is collimated using an AR-coated aspheric lens, and the collimated UV beam is then directed toward a DMD chip at a 24° angle of incidence (corresponding to twice the tilting angle of the DMD mirrors). The image of the DMD chip is then relayed onto the conjugate of the sample plane at the backport of the microscope through a 4f imaging system (f1 = f2 = 125 mm UV fused silica bi-convex lenses, AR-coated). This intermediate image is then relayed onto the sample plane by a tube lens and the objective. To combine DMD-UV illumination with TIRF illumination, a 470-nm dichroic is placed after f2 within a custom backport assembly (Cairn). To offer a second illumination wavelength for 450-nm optogenetic stimulation, our design also contains a second 450-nm collimated LED at the symmetric −24° angle. This LED can be exchanged for any other LED to provide epifluorescence imaging. (B and C) Enhanced fibrinogen micropatterning efficiency depends on the buffer used. (B) PLL-PEG–coated glass was processed for LIMAP patterning with identical UV exposure, micropattern shape, and photoinitiator concentration (50 mM BBTB in 0.1 M sodium bicarbonate, pH 8.3). Fibrinogen-Alexa546 (50 µg/ml) was then adsorbed onto the UV-activated surface in either PBS or carbonate buffer. After washing, red fluorescence of the patterns was imaged by TIRFM using identical settings. (C) Quantification of the effects seen in B (average selectivity ± SEM; see Materials and methods). Fibrinogen quantitatively patterns better in carbonate buffer. Statistics were performed using a Mann–Whitney rank-sum test. n, number of patterns measured. (D–F) Advantages of fibrinogen for multiplexed micropatterning. (D) Scheme illustrating the different steps for sequential multiplexed micropatterning of two fibrinogens labeled with different fluorophores (ATTO488 and Alexa647). (E) Multiplexed micropatterning of fibrinogen-ATTO488 and Alexa647 (50 µg/ml) using the scheme depicted in D onto PLL-PEG–coated glass. Note that there is high specificity of the fibrinogen for their specific pattern and minimum overlap among the two fluorescent fibrinogens. In addition, binding of fibrinogen to unexposed PLL-PEG is minimal, down to a punctate, single molecule–like level (orange arrowheads). (F) Quantification of the effects seen in E: the fluorescence of each Fibrinogen was measured on the two patterns and normalized to the fluorescence of their intended pattern (i.e., first pattern for Alexa647 and second for ATTO488; mean ± SEM). Statistics were performed using a Kruskal–Wallis test followed by a Dunn post hoc test (P < 0.0001). n, number of patterns measured. Note that the vertical scale of the graph is split to better appreciate the minute amounts of fibrinogen deposited onto the nonintended patterns or the unpatterned PLL-PEG area. Scale bars, 10 µm. n.s., not significant.

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