Adequate supply of oxygen is essential for the normal function of cells, and microcirculation plays a critical role in this process. Sufficient oxygen delivery depends on both convective and diffusive components of transport. In the present study, a multispectral imaging approach was used to assess SO2 in an intravital microscopic model. Experiments were performed using the dorsal skinfold chamber in mice. An intravital microscope was modified for epi- and transillumination by a light guide from a monochromatizing device (TILL-Photonics). This optical setup allowed generation and recording of stacks of images at preselected wavelength ranges and exposure times. For the calculation of SO2 values from these lambda-stacks a dedicated software was developed, generating 2D images of oxygen saturation based on haemoglobin spectrophotometry.In larger venules (diameter>15 mm) flow regions with different saturation levels, "SO2-streamlines", could be observed. The two main findings were: (i) streamlines extended over long distances, up to >1mm; (ii) SO2 levels on the arterial side of venules were higher in comparison to the opposite side. The data indicate, that intravascular diffusion in venules is too slow to generate effective SO2 equilibration. The higher SO2 values in venules at the side directed towards the corresponding arteriole may be due to either intervascular diffusion or the presence of arterio-venous shunts.