Loss Of Autofluorescence Research Articles

Changes in chalazal cell walls and in the peroxidase enzymes of the crease region during grain development in barley.

In an investigation of the role of peroxidase enzymes in the differentiation of the tissues of the crease region of barley, plants of winter barley cv. Halcyon were grown from anthesis onwards in controlled conditions at a constant temperature of 16 degrees C. Four ears were harvested at 2-d intervals from 6 d after anthesis (daa) until 50 daa. Grains from mid-ear were used for (i) fresh and dry weight determinations, (ii) extraction of crease tissue for the determination of peroxidase activity and for the separation of isozymes of peroxidase by isoelectric focusing (IEF) and (iii) detection of lignin and suberin in the tissues of the crease using autofluorescence and cytochemistry. Peroxidase activity was located histochemically in the crease tissue of cv. Chariot. Scanning electron microscopy studies were carried out on developing grains of cv. Blenheim. Maximum grain water content was achieved at 14 daa. Lignin and suberin were detected in the walls of the chalazal cells from 18 daa onwards. No changes in the staining of chalazal cell walls were detected at the end of grain filling (32 daa), but loss of autofluorescence and staining were observed at 42 daa, just prior to the final, rapid phase of grain dehydration. Peroxidase activity per fresh weight of crease tissue was high at 6 daa and low at 22 daa. It was also low between 32 and 40 daa, but it rose again from 42 daa onwards. IEF demonstrated that both anionic and cationic isozymes of peroxidase were present in crease tissue, the pattern of bands showing some marked changes during the course of grain development.

Autofluorescence characteristics of lipofuscin components in different forms of late senile macular degeneration

Lipofuscin is the main fluorophore of the human fundus. Because lipofuscin is the result of the accumulation of metabolic debris in pigmentepithelial cells (RPE), the autofluorescence can be interpreted as a clinical sign for the metabolic activity of the RPE. In order to get informations of RPE-function in different types of late AMD, the autofluorescence patterns in patients with late AMD were analyzed. A prospective examination of the fundus-autofluorescence of 64 eyes of 52 patients with different types of late AMD was performed using a confocal scanning-laser-opthalmoscope. The autofluorescence images were categorized in respect to the type of late AMD according to the opthalmoscopic and fluoresceine-angiographic findings. Reduced autofluorescence was found in the centre of occult (78.6%) and classic (100%) choroidal neovascularisations (NV) as well as in the occult NV of RPE detachments. A loss of autofluorescence was related to the RPE free area of RPE-tears (100%) and to RPE-atrophy (88.9%) with sometimes increased autofluorescence at the rim. Increased autofluorescence could be seen at the surface of RPE-detachments (71.4%), in the area of the shrink age of RPE in RPE-tears (100%) as well as at RPE-proliferations in small occult NV (100%). Disciforme scars showed variable patterns of autofluorescence. The autofluorescence of the RPE can be analyzed clinically with the described method. Different patterns of autofluorescence could be revealed in different types of late AMD. Increased autofluorescence was found in lesions with proliferative or phagocytotic metabolic activity of the RPE like RPE-detachments, shrinked RPE in RPE-tears or occult NV with RPE-proliferations. The reduced autofluorescence in occult or classical choroidal NV can be interpreted as a sign of decompensation of the RPE and was also seen in areas with RPE-loss.

Fixation, Counting, and Manipulation of Heterotrophic Nanoflagellates

Quantitative effects of several fixatives on heterotrophic nanoflagellates (HNAN) and phototrophic nanoflagellates (PNAN) were investigated by hemacytometer and epifluorescence counting techniques. Counts of Monas sp. cultures before and after fixation with unbuffered 0.3% glutaraldehyde and 5% formaldehyde showed no loss of cells during fixation, and cell concentrations remained constant for several weeks after fixation. Buffering of fixatives with borax caused severe losses, up to 100% within 2 h. Field samples from Lake Vechten showed no decline of HNAN and total nanoflagellate concentrations for at least 1 week after fixation with 5% formaldehyde and with 1% glutaraldehyde. With 1% glutaraldehyde, the chlorophyll autofluorescence of PNAN was much brighter than with 5% formaldehyde, although it was lost after a few days and thus limited the storage time of samples. However, when primulin-stained slides were prepared soon after fixation and stored at -30 degrees C, the loss of autofluorescence was prevented and PNAN and HNAN concentrations were stable for at least 16 weeks. Effects of filtration and centrifugation on HNAN were also studied. Filtration vacuum could not exceed 3 kPa since 10 kPa already caused losses of 15 to 20%. Similar losses were caused by centrifugation, even at low speed (500 x g).

Histochemical Reactions, Autofluorescence, and Rumen Microbial Degradation of Tissues in Untreated and Delignified Bermudagrass Stems1

Low digestibility of stems is due to large proportions of lignified cell walls, but lignified tissues vary in resistance to microbial degradation. The objective of the research was to characterize lignified tissues histologically and by autofluorescence, and to examine the influence of delignification with potassium permanganate (PM) on the histology, autofluorescence, and tissue degradation by rumen microorganisms in mature internodes of ‘Coastal’ bermudagrass [Cynodon dactylon (L.) Pers.]. Cell walls of epidermis, sclerenchyma ring, and vascular xylem gave a positive lignin test with acid phloroglucinol (AP+), while those the parenchyma gave a positive reaction to the chlorine‐sulfite test (CS+). The maximum of the emission spectrum (Emax) of the autofluorescence after excitation at 365 nm was 452 to 455 nm for the sclerenchyma ring and about 460 nm for parenchyma. The intensity of the autofluorescence for the three internodes averaged 1.97 mV for the sclerenchyma ring, 1.50 mV for the middle parenchyma, and 1.27 mV for parenchyma cell walls nearer the stem center. Delignification resulted in lack of a histochemical reaction in parenchyma, but an AP+ reaction still occurred in epidermis, sclerenchyma ring, and xylem. Delignification reduced the Emax tO 450nm and the intensity to 1.28 mV for AP+ tissues and resulted in a total loss of autofluorescence in parenchyma. Treatment with PM resulted in almost total microbial degradation of the parenchyma, but AP + tissues were not degraded in untreated or PM‐treated stem sections. Results indicate that variations exist in the retention of phenolic compounds in cell walls upon oxidation with PM, and these variations relate to quality differences in forage fiber.