2.3. Fluorescence Microscopy and Software
Living cells bound to chemical spots were imaged using a Nikon TE2000 epifluorescence microscope equipped with a 75 W Xenon light source and an Orca-ER (Hamamatsu, Bridgewater, NJ, USA) camera [25,26]. Two custom filter sets (Chroma Technology Corporation, Brattleboro, VT, USA) composed of 650 ± 22 nm and 750 ± 25 nm excitation filters, 675 nm and 785 nm dichroic mirrors, and 710 ± 25 nm and 810 ± 20 nm emission filters were, respectively, used to detect ESNF10 (700 nm, pseudo-colored in red) and IR786 (800 nm, pseudo-colored in lime green) emission. For high-throughput imaging of microarrays, we have previously developed an automated microscope stage and software [21]. The complete scanning time for one microarray slide containing 5076 spots was approximately 2 h (1 s per spot plus stage movement time) using the automated microscope. IPLab 3.6 software (Nikon Inc., Melville, NY, USA) and ImageJ 1.45q (NIH, Bethesda, MD, USA) were used for normalization and autosegmentation of the fluorescence intensity of each spot. Sequential procedures for scoring were defined through region-of-interest (ROI) selection, static thresholding, binary image, and auto-counting. Data plotting was performed using Prism version 4.0a software (GraphPad, San Diego, CA, USA) and Microsoft Excel (Redmond, WA, USA).