Dimetrodon sp. femur, jaw, teeth, and rib bones were collected at N.E. Frederick, OK, and sectioned for histological examination. This Oklahoma Museum of Natural History site has yielded well-preserved Paleozoic fossils for decades. All bones were heavily infiltrated with calcite and other minerals, yet a reddish-brown mass obstructed many vascular channels. This reddish-brown mass reacted to 374 nm UV illumination by emitting bright blue autofluorescence. This was true for every specimen and indicates a heavy presence of iron. We conclude that the iron in the canals is the crystallized remains of blood that clotted during an asphyxiation event that killed the animal. Remarkably similar reddish-brown obstructive material has also been observed in other histological studies performed on Dimetrodon bones. We conclude that these are also the remains of blood clots and simply await examination under UV fluorescence.

Remarkable discoveries such as condensed chromatin in duckbill dinosaur cartilage and newly presented discoveries of dinosaur vascular veins, venule valves, and nerve fibers (this report) warrant study of global dinosaur remains for preserved soft tissue (dST). Dinosaur osteocytes feature dendritic projections (filipodia) of lengths up to 18 µm, while osteocytes collected from a Triceratops horn have lengths of 25 µm or longer. While preservation methods for dST involve the degradation of sugars into glycation end products and the employment of highly oxidative hydroxyl radicals to “fix” tissues, we note that lengthy and narrow osteocyte filipodia show no signs of hydroxyl radical infiltration into the lacuna-canalicular network. Moreover, our ultraviolet fluorescence (UVFL) study of Triceratops horn, rib, vertebra, and frill thin sections shows extensive clotting in most vessel canals, probably as a result of asphyxia while drowning. Further UVFL autofluorescence study of dinosaur bone sections is vital for characterization of dinosaur blood clots.

Angiostrongylus cantonensis is a pathogenic nematode and the cause of neuroangiostrongyliasis, an eosinophilic meningitis more commonly known as rat lungworm disease. Transmission is thought to be primarily due to ingestion of infective third stage larvae (L3) in gastropods, on produce, or in contaminated water. The gold standard to determine the effects of physical and chemical treatments on the infectivity of A. cantonensis L3 larvae is to infect rodents with treated L3 larvae and monitor for infection, but animal studies are laborious and expensive and also raise ethical concerns. This study demonstrates propidium iodide (PI) to be a reliable marker of parasite death and loss of infective potential without adversely affecting the development and future reproduction of live A. cantonensis larvae. PI staining allows evaluation of the efficacy of test substances in vitro, an improvement upon the use of lack of motility as an indicator of death. Some potential applications of this assay include determining the effectiveness of various anthelmintics, vegetable washes, electromagnetic radiation and other treatments intended to kill larvae in the prevention and treatment of neuroangiostrongyliasis.

Resilin is a soft and elastic protein, which is found in many exoskeletal structures of arthropods. Proteins with similar chemical properties have been described for other invertebrates including monogenean fish parasites. However, for the latter taxon no clear microscopic evidence for a resilin-like protein has been shown so far. Here, we present the results of detailed microscopic analyses of the clamp sclerites (attachment devices) of the monogenean Diplozoon paradoxum. Toluidine blue, which is known to stain resilin, selectively stained the material in the clamp sclerites. In addition, when exposed to UV light, this material exhibited an intense blue autofluorescence. The emission spectrum of this autofluorescence has its maximum at 424 nm and is nearly identical to emission spectra of blue autofluorescences observed in 2 well-studied structures containing high proportions of resilin in the exoskeleton of the locust Schistocerca gregaria. The results strongly indicate that the sclerite material of D. paradoxum contains a resilin-like protein. The presence of such a protein likely enhances the attachment efficiency of the clamp sclerites and increases their lifetime.

Improved understanding of the occurrence and spatio-temporal distribution of bivalve larvae holds significant benefits for ecological studies, shellfisheries management and aquaculture. Morphological methods for identification have proved difficult to develop because of the small size of these larvae and similarities in their shape and colour. Molecular methods based on DNA extraction can confirm the presence of a species in a plankton sample, but without sample sorting and individual larval analysis, provide no estimate of larval abundance and are incapable of providing an estimate of larval growth rate. Fluorescence in situ hybridization (FISH) using species-specific DNA probes has the potential to resolve these issues. However, utilization of this technique is constrained by the strong autofluorescence, common in marine larvae. Here we evaluate the effect of eight different fixatives on the autofluorescence intensity of bivalve larvae using fluorescein isothiocyanate (FITC) and Cy3 filters. In addition, fifteen autofluorescence reduction treatments were evaluated and their compatibility with FISH assessed. Relative to fresh larvae, chemically fixed larvae had significantly higher autofluorescence in both filter sets. Larvae preserved by freezing at –80°C exhibited no significant increase in autofluorescence over a 3-year period. Autofluorescence levels were generally lower with the FITC filter set than the Cy3 filter set. For archived larvae preserved in modified saline ethanol and exhibiting fixative-induced autofluorescence, the autofluorescence intensity could be reduced to 20–30% with saturated Sudan Black B and to 30–40% with Chemicon™. Both of these autofluorescence reduction treatments were compatible with subsequent FISH protocols using a FITC-labelled probe.

Use of deep ultraviolet (DUV, below 350 nm) fluorescence opens up new possibilities in biology because it does not need external specific probes or labeling but instead allows use of the intrinsic fluorescence that exists for many biomolecules when excited in this wavelength range. Indeed, observation of label free biomolecules or active drugs ensures that the label will not modify the biolocalization or any of its properties. In the past, it has not been easy to accomplish DUV fluorescence imaging due to limited sources and to microscope optics. Two worlds were coexisting: the spectrofluorometric measurements with full spectrum information with DUV excitation, which lacked high-resolution localization, and the microscopic world with very good spatial resolution but poor spectral resolution for which the wavelength range was limited to 350 nm. To combine the advantages of both worlds, we have developed a DUV fluorescence microscope for cell biology coupled to a synchrotron beamline, providing fine tunable excitation from 180 to 600 nm and full spectrum acquired on each point of the image, to study DUV excited fluorescence emitted from nanovolumes directly inside live cells or tissue biopsies.

Cellular autofluorescence, though ubiquitous when imaging cells and tissues, is often assumed to be small in comparison to the signal of interest. Uniform estimates of autofluorescence intensity obtained from separate control specimens are commonly employed to correct for autofluorescence. While these may be sufficient for high signal-to-background applications, improvements in detector and probe technologies and introduction of spectral imaging microscopes have increased the sensitivity of fluorescence imaging methods, exposing the possibility of effectively probing the low signal-to-background regime. With spectral imaging, reliable monitoring of signals near or even below the noise levels of the microscope is possible if compensation for autofluorescence and background signals can be performed accurately. We demonstrate the importance of accurate autofluorescence modeling and the utility of spectral imaging and multivariate analysis methods using a case study focusing on fluorescence confocal spectral imaging of host-pathogen interactions. In this application fluorescent proteins are produced when Francisella novicida invade host macrophage cells. The resulting analyte signal is spectrally overlapped and typically weaker than the cellular autofluorescence. In addition to discussing the advantages of spectral imaging for following pathogen invasion, we present the spectral properties and cellular origin of macrophage autofluorescence.

The extent of arbuscular mycorrhizal colonization was assessed in 10 field-collected plant species, representing three annual forbs, three perennial forbs, three perennial grasses and one annual grass. Each root system of each plant was split into four portions, and for each portion, mycorrhizal structures were revealed with epifluorescence microscopy (under which only arbuscules are generally visible) and three commonly used stains (Chlorazol Black E, Acid Fuchsin and Trypan Blue). The aim of the study was not to evaluate the efficacy of each method, but to compare results obtained by each under standard laboratory conditions. The recorded colonization levels of arbuscules, total arbuscular mycorrhizal fungal material and total fungal (arbuscular mycorrhizal+non-arbuscular mycorrhizal) material differed significantly between visualization methods in a number of species. However, there were also interactions between stain and plant species, indicating that the performance of a stain is dependent on the plant species being examined. In some cases (e.g. Plantago lanceolata), each visualization method produced the same colonization level, while in others (e.g. Dactylis glomerata), each method gave a different result. These data therefore suggest that the level of mycorrhizal colonization recorded in any particular plant species at a particular time is dependent on the technique employed.