Selective Staining by the Fluorochrome, 5,5'-Diphenyl-9-ethyl-DiOC2(3). I. Physicochemical Studies of Dye-Dye and Dye-Tissue Interactions

The 5,5'-diphenyl-9-ethyl-oxacarbocyanine (5,5'-diphenyl-9-ethyl-DiOC₂(3); CD) has properties suitable for histological investigations including the spectral range for absorption and fluorescence emission, the values of the corresponding molar coefficients and fluorescence quantum yield. Furthermore, CD remains relatively unchanged over the entire pH range and interacts with protein β-sheets. The latter fact is detectable spectroscopically as a bathochromic shift. In water-containing media such as the histological stain and washes, CD exists as a monomer, a dimer and in two aggregated states. These differ in their binding affinity to tissue sections, in their solubility in water, alcohol and water/alcohol mixtures, and in their UV/VIS absorption and fluorescence emission. The ratio of the various CD states and the contrast of selectively stained tissue areas can be controlled via the staining conditions and the sequence of the washes. Furthermore, mounting in a xylene-based medium produces a solvatochromic spectral shift of the CD monomer, which leads to a marked elevation in phase contrast.