Collagen I is the major structural protein in mammals where it exhibits highly organized fibrillar distributions in connective tissues. In vitro, acidic solutions of collagen I display lyotropic liquid crystal organization. These concentrated organized liquid phases can be stabilized by a pH increase to generate in vitro fibrillar matrices with specific organization. The aim of this work is to understand the mechanisms responsible for liquid crystal chirality at acidic pH in order to guide the synthesis of collagen matrices reproducing the great diversity of organizations found in biological tissues. For this purpose, we quantitatively analyze collagen liquid crystal organization by use of multiphoton microscopy, combining fluorescence and second harmonic generation contrasts. The concentration of the isotropic to liquid crystal phase transition and the evolution of the half pitch of the helical phase with collagen concentration are reported in five physico-chemical conditions using hydrochloric and acetic acids at different pHs and ionic strengths. A new phase transition is observed in highly concentrated solutions ranging from 90 mg ml-1 to 300 mg ml-1 depending on the solvent. Our results bring new quantitative information on collagen chemical physics and further substantiate the on-going analysis of the driving parameters generating twists in liquid crystals. These findings could be advantageously exploited to develop new strategies and protocols for tissue engineering. This is crucial for fundamental studies of cell behavior in biomimetic three-dimensional environments and for medical and pharmaceutical applications. Cop. The Royal Society of Chemistry 2011.