Microvascular permeability is a serious complication of systemic inflammation in critically ill patients; yet, no direct techniques exist to quantify this in vivo. To overcome this limitation, we investigated the use of multiphoton microscopy to evaluate fluorescent macromolecular gradients in the eye. Following the induction of systemic inflammation in a CD1 mouse, a bolus of high (250 KD FITC-dextran) and low (70 KD rhodamine-dextran) molecular weight fluorescent macromolecules was injected via the tail vein. The anesthetized mouse was positioned in such a way that different microvessels in the eye could be imaged directly using an upright microscope. The fluorophores were simultaneously excited at 840nm and a series of images including a spectral scan (480 to 680nm), an xt line scan (96 lines) and an x,y,z image stack were collected from the iris, cornea and limbal plexus at one hour intervals for four hours. A simple fluorescent gradient across the vessel wall was used as an index of microvascular permeability. In all microvessels, the LMW dye was more permeable. We found that the fluorescent gradient increased dramatically in the limbal plexus up to three hours then declined. This may indicate that circulating fluid pooled near the limbal plexus. Consistent with the thick walls and tight junctions of the iris microvessels, no significant fluorescent gradients were detected in this area. The cornea, containing a collagen filled stroma layer, was found to have both lateral and perpendicular fluorescent gradients. This work demonstrates that inflammation causes differential microvascular permeability in the mouse eye.