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 -60°C 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
0.1°C. 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.
The structural remodeling of extracellular matrix proteins in peripheral lung region is an important feature in chronic
obstructive pulmonary disease (COPD). Multiphoton microscopy is capable of inducing specific second harmonic
generation (SHG) signal from non-centrosymmetric structural proteins such as fibrillar collagens. In this study, SHG
microscopy was used to examine structural remodeling of the fibrillar collagens in human lungs undergoing
emphysematous destruction (n=2). The SHG signals originating from these diseased lung thin sections from base to apex
(n=16) were captured simultaneously in both forward and backward directions. We found that the SHG images detected
in the forward direction showed well-developed and well-structured thick collagen fibers while the SHG images detected
in the backward direction showed striking different morphological features which included the diffused pattern of
forward detected structures plus other forms of collagen structures. Comparison of these images with the wellestablished
immunohistochemical staining indicated that the structures detected in the forward direction are primarily the
thick collagen type I fibers and the structures identified in the backward direction are diffusive structures of forward
detected collagen type I plus collagen type III. In conclusion, we here demonstrate the feasibility of SHG microscopy in
differentiating fibrillar collagen subtypes and understanding their remodeling in diseased lung tissues.