Multiphoton microscopy based cryo-imaging of inflated frozen human lung sections at -60OC in healthy and COPD lungs

Lung is a complex gas exchanger with interfacial area (where the gas exchange takes place) is about the size of a tennis court. Respiratory function is linked to the biomechanical stability of the gas exchange or alveolar regions which directly depends on the spatial distributions of the extracellular matrix fibers such fibrillar collagens and elastin fibers. It is very important to visualize and quantify these fibers at their native and inflated conditions to have correct morphometric information on differences between control and diseased states. This can be only achieved in the ex vivo states by imaging directly frozen lung specimens inflated to total lung capacity. Multiphoton microscopy, which uses ultra-short infrared laser pulses as the excitation source, produces multiphoton excitation fluorescence (MPEF) signals from endogenously fluorescent proteins (e.g. elastin) and induces specific second harmonic generation (SHG) signals from non-centrosymmetric proteins such as fibrillar collagens in fresh human lung tissues [J. Struct. Biol. (2010)171,189-196]. Here we report for the first time 3D image data obtained directly from thick frozen inflated lung specimens (∼0.7- 1.0 millimeter thick) visualized at -60OC without prior fixation or staining in healthy and diseased states. Lung specimens donated for transplantation and released for research when no appropriate recipient was identified served as controls, and diseased lung specimens donated for research by patients receiving lung transplantation for very severe COPD (n=4) were prepared as previously described [N. Engl. J. Med. (2011) 201, 1567]. Lung slices evenly spaced between apex and base were examined using multiphoton microscopy while maintained at -60°C using a temperature controlled cold stage with a temperature resolution of 0.1OC. Infrared femto-second laser pulses tuned to 880nm, dry microscopic objectives, and non-de-scanned detectors/spectrophotometer located in the reflection geometry were used for generating the 3D images/spectral information. We found that this novel imaging approach can provide spatially resolved 3D images with spectral specificities from frozen inflated lungs that are sensitive enough to identity the micro-structural details of fibrillar collagens and elastin fibers in alveolar walls in both healthy and diseased tissues.