Oxadiazon (10 μM) caused rapid, light-dependent membrane damage to cucumber cotyledon discs. Electrolyte leakage was detected within 1 h of exposure to light, as were cytoplasmic vesiculation and breakage of the tonoplast and plasmalemma. The ultrastructure of chloroplasts was not affected until the cytoplasm was dispersed. Photosynthetic inhibitors had no effect on activity and, after a period of dark incubation with oxadiazon, there was little effect of temperature on the light-caused membrane destruction. Porphyrin synthesis inhibitors (gabaculine and 4,6-dioxoheptanoic acid) almost completely prevented the herbicidal activity of oxadiazon. Oxadiazon treatment caused accumulation of protoporphyrin IX, a photodynamic pigment. Oxadiazon caused physiological effects on cucumber cotyledons that were virtually identical to those of p-nitro-diphenyl ether herbicides like acifluorfen and its methyl ester, which have recently been shown to also cause protoporphyrin IX accumulation.

Fluorescence images present low signal-to-noise ratio (SNR), are corrupted by a type of multiplicative noise with Poisson distribution, and are affected by a time intensity decay due to photoblinking and photobleaching (PBPB) effects. The noise and the PBPB effects together make long-term biological observation very difficult. Here, a theoretical model based on the underlying quantum mechanic physics theory of the observation process associated with this type of image is presented and the common empirical weighted sum of two decaying exponentials is derived from the model. Improvement in the SNR obtained in denoising when the proposed method is used is particularly important in the last images of the sequence where temporal correlation is used to recover information that is sometimes faded and therefore useless from a visual inspection point of view. The proposed PBPB model is included in a Bayesian denoising algorithm previously proposed by the authors. Experiments with synthetic and real data are presented to validate the PBPB model and to illustrate the effectiveness of the model in denoising and reconstruction results.

We report our efforts in identifying optimal scanning laser microscope parameters to study cells in three-dimensional culture. For this purpose we studied contrast of extracellular matrix (ECM) mimics, as well as signal attenuation, and bleaching of red and green fluorescent protein labeled cells. Confocal backscattering, second harmonic generation (SHG), and autofluorescence were sources of contrast in ECM mimics. All common ECM mimics exhibit contrast observable with confocal reflectance microscopy. SHG imaging on collagen I based hydrogels provides high contrast and good optical penetration depth. Agarose is a useful embedding medium because it allows for large optical penetration and exhibits minimal autofluorescence. We labeled breast cancer cells' outline with DsRed2 and nucleus with enhanced green fluorescent protein (eGFP). We observed significant difference both for the bleaching rates of eGFP and DsRed2 where bleaching is strongest during two-photon excitation (TPE) and smallest during confocal imaging. But for eGFP the bleaching rate difference is smaller than for DsRed2. After a few hundred microns depth in a collagen I hydrogel, TPE fluorescence of DsRed2 becomes twice as strong compared to confocal imaging. In fibrin and agarose gels, the imaging depth will need to be beyond 1 mm to notice a TPE advantage.

The lateral diffusion (DL) properties of the fluorescent lipid probe 5-N (octadecanoyl) aminofluorescein (AF18) inserted into the surface of muscle-stage larvae of Trichinella spiralis were investigated by fluorescence recovery after photobleaching. AF18 was not free to diffuse laterally in dormant larvae, and this remained unchanged after larval activation in vitro with trypsin and bile. However, a significant increase in surface fluidity of the probe was demonstrated (%R = 74·5; DL = 11·5 × 10−9 cm2/sec) when larvae invaded intestinal epithelial tissue following oral infection of mice. Membrane-permeant photoactivatable caged cyclic AMP was used to analyse the putative mechanism responsible for this increase in lateral diffusion in the parasite surface. Although incubation of larvae with 1–50 μM caged cAMP had no effect on surface fluidity, incubation with 100 μM caged cAMP induced a substantial increase in the lateral mobility of AF18 (%R = 64·3; DL = 8·3 × 10−11 cm2/sec) immediately following photo-activation of the caged messenger. This induced fluidity, however, was transient and the larval surface reverted to immobility within 15 min. These observations constitute the first reported measurement of the fluid properties of the surface of intracellular parasites, the first demonstration of the parasite surface fluidity altering as a result of host cell invasion and the first indication of a mechanism underlying changes in surface fluidity in parasitic helminths.