Cryopreserved valve allografts offer superior hemodynamic performance in ventricular outflow reconstruction. Unfortunately, structural degeneration of the valve leaflets limits long-term durability. The performance and durability of any valve are dependent on the exquisitely complex tissue structure of the valve and annulus. Any disruption of the extracellular matrix structure will change the biomechanics of the tissue and may alter the function of any remaining viable cells. Schenke-Layland and colleagues [1Schenke-Layland K. Madershahian N. Riemann I. et al.Impact of cryopreservation on extracellular matrix structures of heart valve leaflets.Ann Thorac Surg. 2006; 81: 918-927Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar] have made a step forward in our understanding of the structural deterioration of cryopreserved tissue. In their analysis of porcine pulmonary valves, standard biochemical assays suggested a preservation of the elastin and collagen content after cryopreservation. However, closer examination by multiphoton autofluorescence imaging demonstrates serious alteration in the structural arrangement of proteins. Interestingly, this disruption is most pronounced in the ventricularis layer in which both immunohistochemistry and nonlinear optical tomography demonstrated a loss of collagen, particularly type I. The cells in this layer were shown to be damaged (Fig 5) [1Schenke-Layland K. Madershahian N. Riemann I. et al.Impact of cryopreservation on extracellular matrix structures of heart valve leaflets.Ann Thorac Surg. 2006; 81: 918-927Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar]. The mechanisms of this local valve structural alteration after cryopreservation need to be better understood, but is probably related to an activation of metalloproteinases (MMPs) upon thawing [2Kano M. Masuda Y. Tominaga T. et al.Collagen synthesis and collagenase activity of cryopreserved heart valves.J Thorac Cardiovasc Surg. 2001; 122: 706-711Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar], and disruption of normal extracellular matrix metabolism by the valve interstitial cells [3Walker G.A. Masters K.S. Shah D.N. Anseth K.S. Leinwand L.A. Valvular myofibroblast activation by transforming growth factor-beta implications for pathological extracellular matrix remodeling in heart valve disease.Circ Res. 2004; 95: 253-260Crossref PubMed Scopus (312) Google Scholar], a function shown to be impaired in cryopreserved tissue [4Kneebone J.M. Lupinetti F.M. Procollagen synthesis by fresh and cryopreserved rat pulmonary valve grafts.J Thorac Cardiovasc Surg. 2000; 120: 596-603Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar]. Side specific cell phenotypes and differences in extracellular matrix organization may account for the heterogeneity in extracellular matrix alteration [5Simmons C.A. Grant G.R. Manduchi E. Davies P.F. Spatial heterogeneity of endothelial phenotypes correlates with side-specific vulnerability to calcification in normal porcine aortic valves.Circ Res. 2005; 96: 792-799Crossref PubMed Scopus (209) Google Scholar, 6Scott M.J. Vesely I. Morphology of porcine aortic valve cusp elastin.J Heart Valve Dis. 1996; 5: 464-471PubMed Google Scholar]. In addition, the authors note a loss in collagen crimp, the tendency of unloaded collagen to have a rumpled configuration [7Parry D.A.D. The Molecular and fibrillar structure of collagen and its relationship to the mechanical-properties of connective-tissue.Biophysical Chemistry. 1988; 29: 195-209Crossref PubMed Scopus (203) Google Scholar]. Crimp is a major cause of the characteristic nonlinear mechanical response of soft tissue. Loss of this crimp may be due to a loss of the internal restoring force of the elastin as seen in Figure 5 [1Schenke-Layland K. Madershahian N. Riemann I. et al.Impact of cryopreservation on extracellular matrix structures of heart valve leaflets.Ann Thorac Surg. 2006; 81: 918-927Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar]. The lack of crimp in the thawed cryopreserved valves suggests that the biomechanical properties of the ventricularis layer have been altered; however, no study to date has conducted the detailed biomechanical testing needed to verify this effect. The biological effects of the structural alteration caused by cryopreservation demonstrated in this article now have to be related to the performance and durability of allograft human valves. The consequences of these structural changes to the biomechanics and cell biology of the valve before implantation need to be investigated. Multiphoton tomography is a powerful new tool for determining molecular mechanisms and structural biology. Its applications to cardiovascular research will continue to increase as it can provide supporting evidence for structural deterioration in intact tissue, as shown in this article.