Figure 7. Fabrication of Micron-Scale Ligand Gradients.

LSL provides two methods to create ligand density gradients. The total amount of energy delivered to the ROIs can be varied by changing the number of iterations per ROI as depicted in (A) or by slightly tilting the sample so that the sample slowly leaves the focal plane of the focused laser as depicted in (B). (C) A 10 nm Au film was functionalized with an OEG-terminated alkanethiol and 1×8 μm patterns of the OEG were thermally desorbed with a 532 nm laser focused through a 20×(NA0.8) objective operating at a constant fluence. The bare Au patterns were backfilled with a biotin-terminated alkanethiol, labeled with AF546-conjugated streptavidin, and imaged. The number of iterations per ROI was decreased by a factor of 2 from the left to the center of the image and increased by a factor of 2 from the center to the right of the image to create a symmetrical density gradient using the method depicted in (A). (C) The last column on the right (outlined by dashed yellow line and indicated by the yellow arrows) was created by tilting the sample 1.4° so that the laser slowly went out of focus from the top to bottom using the method depicted in (B). (D) An areal plot of the streptavidin fluorescent intensity shows the gradients created in the × direction across all columns and in the y direction for the last column on the far right in (C) as indicated by the yellow arrows. (E) A line plot of the streptavidin intensity as indicated by the red line in (C). (B) SB=20 μm.