Protamine crosslinking by disulphide (-SS-) bonds in the main factor responsible for the stability of chromatin structure in mammalian spermatozoa. Sperm chromatin containing arginine/cysteine-rich protamines shows a deeply modified cytochemical reactivity (e.g. basophilia) when compared with somatic chromatic. After methanol or ethanol-acetic acid fixation and toluidine blue (TB) staining, most sperm heads in semen smear from human fertile donors exhibited a pale blue (orthochromatic) colour, while a few sperm heads exhibited violet-blue or violet (metachromatic) staining. Smears from infertile patients showed an increased amount of metachromatic sperm nuclei. After reduction of -SS- bonds by dithiothreitol, sperm heads from all smears were metachromatic, suggesting that DNA phosphates then become available for TB stacking. Micro- and macro-spectrophotometric studies confirmed the microscopic colour reaction of sperm nuclei. The ortho-/metachromatic ration seems a useful parameter for evaluation of altered chromatin structure in spermatozoa cells. Taking into account the current interest in complementary staining tests for evaluation of semen quality, the metachromatic TB reaction represents a simple cytochemical approach for detecting sperm chromatin abnormalities based on differences in -SS- crosslinking.

We evaluated a number of lipophilic dyes and fluorochromes, including oxazone and thiazone derivatives of oxazine and thiazine dyes, scintillator agents, a carotenoid and a metal-porphyrin complex for visualization of lipid droplets within aldehyde fixed cultured HeLa and BGC-1 cells. Observation under ultraviolet, blue or green exciting light revealed selective fluorescence of lipid droplets, particularly after treatment with aqueous solutions of Nile blue and brilliant cresyl blue oxazones, toluidine blue thiazone, or propylene glycol solutions of canthaxanthin, ethyl-BAO, and ZnTPyP. Mounting in water was required to maintain the fluorescence of lipids; the use of glycerol, Mowiol or Vectashield was not adequate. The effect of dye structure on staining intensity was assessed with the aid of numerical structure parameters modeling lipophilicity (HI and log P), overall size (MW) and the size of the conjugated system (conjugated bond number; CBN). The best stains for lipid droplets were relatively lipophilic (HI > 4.0, log P > 5.0), of small size overall (MW < 370), with small conjugated systems (CBN < 24), and not significantly amphiphilic. The two hydrophobic-hydrophilic parameters (the classic log P and the hydrophobic index, HI; values calculated by molecular modeling software) were highly correlated; however, HI was a more suitable hydrophobicity index for the dyes studied here.

We discuss a variety of biological targets including generic biomembranes and the membranes of the endoplasmic reticulum, endosomes/lysosomes, Golgi body, mitochondria (outer and inner membranes) and the plasma membrane of usual fluidity. For each target, we discuss the access of probes to the target membrane, probe uptake into the membrane and the mechanism of selectivity of the probe uptake. A statement of the QSAR decision rule that describes the required physicochemical features of probes that enable selective staining also is provided, followed by comments on exceptions and limits. Examples of probes typically used to demonstrate each target structure are noted and decision rule tabulations are provided for probes that localize in particular targets; these tabulations show distribution of probes in the conceptual space defined by the relevant structure parameters ("parameter space"). Some general implications and limitations of the QSAR models for probe targeting are discussed including the roles of certain cell and protocol factors that play significant roles in lipid staining. A case example illustrates the predictive ability of QSAR models. Key limiting values of the head group hydrophilicity parameter associated with membrane-probe interactions are discussed in an appendix.

Simple methods for predicting intercalation or groove binding of dyes and analogous compounds with double stranded DNA are described. The methods are based on a quantitative assessment of the aspect (width to length) ratio of the dyes. The procedures were validated using a set of 38 cationic dyes of varied chemical structures binding to well oriented DNA fibers and assessing binding orientation by linear dichroism and polarized fluorescence. We demonstrated that low aspect ratio dyes bound by intercalation, whereas more rod-like dyes were groove binders. Some problems that result and possible applications are discussed briefly.