Figure 3

Revealing details with MPM and correlation with SLOT data.

(a) Volumetric MPM imaging of the CRISTAL sample. After cutting for histological sections, the remaining CRISTAL block is immersed in a cuvette. The plane surface is positioned parallel to the xy-detection plane of the objective. By scanning ultra-short laser pulses, two-photon (2P) excited fluorescence and SHG are recorded simultaneously. In order to generate large volumetric stacks at high resolution, the CRISTAL sample is translated in the xy-plane for 2D mosaicing and the objective is positioned in the z-direction to measure at deeper layers. Thereby, a volume of 7 mm × 16 mm × 397 μm at subcellular resolution level is captured. (b) 2D MPM mosaic image showing 2P excited fluorescence and SHG (blue) in a complete slice through the accessory lobe on the surface of the CRISTAL block. (c) Corresponding reslices from SLOT data showing the correlation of fluorescence (green) and absorption (red) channel at the boxed regions in b. (d) Enlarged views from b at positions indicated with boxes in c shows the alveolar architecture at subcellular resolution. (e) Overlay of maximum intensity projections (MIP) from SHG and fluorescence channel through whole volume depth of the regions shown in d. (f) MIP of fluorescence channel through the complete volume demonstrating the stability and potential of the long term MPM measurements. The boxed MIP of SHG visualizes collagen fiber structures within the pleura. Scale bars, 1 mm (b,f), 500 μm (c), 100 μm (d,e).