Fig. 4. Imaging of endogenous nonlinear optical signals in the mouse spinal cord.

(A) 2PEF imaging of YFP labeled axons (left), CARS imaging of myelin (middle), and an overlay of both modalities (right). Scale bar = 15 µm. (B) 2PEF of GFP labeled microglia (red) and CARS imaging of myelin (green). Scale bar = 15 µm. A and B reproduced from (Bélanger et al., 2012). (C) 2PEF imaging of YFP labeled axons (green) and THG (grey), which highlights myelin. The magnified image to the right (at location of blue box) shows the THG signal from myelin (arrowheads) wrapping tightly around a YFP labeled axon. Imaging done using 1,040-nm excitation light. Reproduced from (Farrar et al., 2011). (D) THG images taken using 1,320-nm excitation light at the surface of the spinal cord before (left) and 30 min after (right) photothrombotic occlusion of a surface venule. THG contrast is produced both by the myelin and by red blood cells inside the vessel (visible as streaks in the image on the left due to their motion). After the occlusion, only stationary red blood cells (which show a characteristic donut shape) are visible and the nearby myelin has begun to degenerate (unpublished data). (E) (top) 3PEF imaging of a spinal cord capillary labeled with an intravenous injection of FITC-dextran. (bottom) Space-time images from repetitive line scans along the axis of the capillary with THG and 3PEF signals detected simultaneously, showing the mutual exclusivity of these two signals in blood, with red blood cells producing THG while excluding the fluorescent dye that labels the blood plasma (unpublished data).