Performing localized chemical events on surfaces is critical for numerous applications. We earlier invented the microfluidic probe (MFP), which circumvented the need to process samples in closed microchannels by hydrodynamically confining liquids that performed chemistries on surfaces (Juncker et al. Nat. Mater.2005, 4, 622-628). Here we present a new and versatile probe, the vertical MFP (vMFP), which operates in the scanning mode while overcoming earlier challenges that limited the practical implementation of the MFP technology. The key component of the vMFP is the head, a microfluidic device (∼1 cm2 in area) consisting of glass and Si and having microfluidic features fabricated in-plane in the Si layer. The base configuration of the head has two micrometer-size channels that inject/aspirate liquids and terminate at the apex which is ∼1 mm2. In scanning mode, the head is oriented vertically with the apex parallel to the surface with typical spacing of 1-30 μm. Such length scales and using flow rates from nanoliters/second to microliters/second allow chemical events to be performed on surfaces with tens of picoliter quantities of reagents. Before scanning, the head is clipped on a holder for leak-free, low dead volume interface assembly, providing a simple world-to-chip interface. Surfaces are scanned by mounting the holder on a computer-controlled stage having ∼0.1 μm resolution in positioning. We present detailed steps to fabricate vMFP heads having channels with dimensions from 1 μm × 1 μm to 50 μm × 50 μm for liquid localization over areas of 10-10000 μm2. Additionally, advanced design strategies are described to achieve high yield in fabrication and to support a broad range of applications. These include particulate filters, redundant aperture architectures, inclined flow-paths that service apertures, and multiple channels to enable symmetric flow confinement. We also present a method to characterize flow confinement and estimate the distance between the head and the surface by monitoring the evolution of a solution of fluorescently labeled antibody on an activated glass surface. This flow characterization reveals regimes of operation suitable for different surface topographies. We further integrate the dispensing of immersion liquid to the vMFP head for processing surfaces for extended periods of time (∼60 min). The versatility of the vMFP is exemplified by patterning fluorescently labeled proteins, inactivation of cells using sodium hypochlorite, and staining living NIH fibroblasts with Cellomics. These applications are enabled by the compact design of the head, which provides easy access to the surface, simplifies alignment, and enables processing surfaces having dimensions from the micrometer to the centimeter scale and with large topographical variations. We therefore believe that ease-of-operation, reconfigurability, and conservative use of chemicals by the vMFP will lead to its widespread use by microtechnologists and the chemical and biomedical communities. © 2011 American Chemical Society.