Immunohistochemical and ultrastructural evidence of functional organization along the Corydoras paleatus intestine

The Neotropical catfish, Corydoras paleatus (Callichthyidae) is a facultative air‐breathing teleost that makes use of the caudal portion of the intestine as an accessory air‐breathing organ. This portion is highly modified, being well vascularized with capillaries between epithelial cells, which makes it well suited for gas exchange. Instead, the cranial portion is a digestion and absorption site, as it has a typical intestinal epithelium with columnar cells arranged in a single row, villi and less vascularized tunica mucosa. Therefore, the intestine was studied by light and electron microscopy to assess differences between the cranial, middle and caudal portions. To characterize the potential for cell proliferation of this organ, we used anti‐proliferating cell nuclear antigen antibody and anti‐Na⁺K⁺‐ATPase monoclonal antibody to detect the presence of Na⁺/K⁺ pump. In C. paleatus it was observed that cell dynamics showed a decreasing gradient of proliferation in cranio‐caudal direction. Also, the intestine of this catfish is an important organ in ionoregulation: the basolateral Na⁺/K⁺ pump may have an active role, transporting Na⁺ out of the cell while helping to maintain the repose potential and to regulate cellular volume. Microsc. Res. Tech. 79:140–148, 2016. © 2016 Wiley Periodicals, Inc.     

Use of primary cell cultures and intact isolated glandular epithelia for X-ray microanalysis

Changes in the elemental composition of cells during isolation of glandular epithelia were studied by electron probe X-ray microanalysis. Fine chopping of rat submandibular gland followed by enzymatic treatment for 15 min caused marked increases in Na and Cl and a decrease in K concentrations in acinar cells. After enzymatic treatment for 50 min, Na, Cl and K concentrations returned to close to the control level. Mechanical disaggregation of the acinar clumps following enzymatic treatment resulted again in minor increases in Na and Cl and a marked decrease in K concentration. Exposure of isolated acini to cholinergic stimulation in vitro resulted in secretion of Cl and K from the acinar cells. Dissection of the sweat gland from human skin caused a decrease in the K/Na ratio. Incubation of the gland for 30–45 min with collagenase gave rise to a gradual decrease in the K/Na ratio. After mechanical separation of the gland into the secretory coil and reabsorptive duct, a further reduction of the K/Na ratio was seen. However, the duct cells had a much lower K/Na ratio and higher Ca concentration than the coil cells. In primary cultures, the K/Na ratios of the coil and duct cells returned to the in situ level. The elemental composition of sweat gland cells incubated in collagenase-containing medium was no different from that in cells incubated in collagenase-free medium. In the intact collagenase-isolated tissue, Cl^? secretion in the coil was elicited by carbachol but not by cAMP, whereas in the duct cells the reverse was the case. In primary cell cultures, Cl^? efflux in both coil and duct cells could be elicited by both carbachol and cAMP. In conclusion, although changes in elemental composition of gland cells during the isolation procedure occur, physiological responses can be detected. When primary cell cultures are used, it should be borne in mind that cultured cells may have physiological properties different from those of the intact tissue.     

Compact Design for a Square Wave Excitation Contactless Conductivity Detector: Determination of Metal Cations in Environmental Water Samples

Abstract

A compact and inexpensive contactless conductivity detection (CCD) based on a square wave excitation voltage, was implemented by integrating the function generator, detective cell, and detective circuit on a printed circuit board with a size of 100 mm length and 50 mm width, and it exhibited excellent sensitivity at the optimal frequency of 198 kHz. The separation for 13 metal ions was demonstrated by optimizing some most significant concentration factors, such as the concentration of 2‐(N‐morpholino) ethanesulfonic acid/DL‐histidine (MES/His), cetyltrimethylammonium bromide (CTAB), and α‐hydroxyisobutyric acid (HIBA). Under the optimized condition of 15 mM MES/His, 0.02 mM CTAB, and 0.5 mM HIBA, the detection limits were in the range of 0.9～5 µM for K⁺, Ba²⁺, Ca²⁺, Na⁺, Mg²⁺, Mn²⁺, and Li⁺; 7～25 µM for Pb²⁺, Cd²⁺, Zn²⁺, Co²⁺, Cu²⁺, and Ni²⁺. This CCD was further used to determine the metal cations in the real samples, such as tap water and low pH galvanic bath rinse water, and the results demonstrated that this CCD system was feasible for analysis of ions in an environmental sample.     

Sodium localization in Suaeda maritima leaf cells using zinc uranyl acetate precipitation

The use of zinc uranyl acetate as a sodium precipitant was investigated during conventional fixation and freeze-substitution preparations; it was superior to antimonate reagents insofar as it only precipitated Na⁺ and K⁺ (Ca²⁺ and Mg²⁺ were not detected) and could be used in conjunction with freeze-substitution in acetone.     

Ion microscopy imaging of various pyrimidic nucleotides and analogs in the nuclei of cultured tumor cells

New applications for ion microscopy are presented. This method has been used primarily to detect mineral elements (K⁺, Na⁺, AL⁺). It also can be used to detect organic molecules containing halogen atoms and radioactive isotopes. ¹⁴C-nucleotides and halogenated pyrimidic nucleotides and analogs were employed in this study. The various images obtained were correlated with the mechanism of action of these compounds, thus opening new lines of research.     

Freeze-substitution and the preservation of diffusible ions

Freeze-substitution of biological material in pure acetone followed by low-temperature embedding in the Lowicryls K11M and HM23 yields stable preparations well suited for sectioning and subsequent morphological and microanalytical studies. Transmission electron microscopy of dry-cut sections shows that diffusible cellular thallium ions (Tl⁺) of Tl⁺-loaded muscle are localized at similar protein sites in freeze-substituted as in frozen-hydrated preparations. A comparison of X-ray micro-analytical data obtained from freeze-dried cryosections and sections of freeze-substituted normal (potassium-containing) muscle shows that K⁺ ion retention in the freeze-substituted sample is highly dependent on the freeze-substitution procedure used; so far, in the best case, about 67% of the cellular K⁺ is retained after freeze-substitution in pure acetone and low-temperature embedding. It is concluded that the retention of diffusible cellular ions is dependent on their interactions with cellular macromolecules during the preparative steps and that ion retention may be increased by further optimizing freeze-substitution and low-temperature embedding.     

The intracellular distribution of ions and water in rat liver and heart muscle

Local dry mass concentrations of intracellular compartments in rat heart muscle and liver cells were estimated by quantitative electron microscopy and X-ray microanalysis of ultrathin frozen-dried cryosections. The results were used to calculate elemental concentrations per litre of compartment water from the X-ray microanalytical data. Water fractions were between 80.3 ± 1.3% of wet weight in the decondensed chromatin and only 45.1 ± 1.7% in mitochondria of liver cells. The lowest water fraction in heart muscle cells was also found in mitochondria. The ionic concentrations found in the cytoplasm of liver cells and in the myofibrils are in accord with the electroneutrality rule and in osmotic equilibrium with the extracellular concentrations. The concentrations of Na, K, Cl and P both in the cytoplasm and in the regions of decondensed chromatin within the nuclei were found to be equal. However, in regions of condensed chromatin K⁺ concentrations were found to be much higher than expected for a Donnan distribution of ions free in solution. Most probably the activity coefficient for K⁺ is lower in the condensed chromatin than in the decondensed or in the cytoplasm. The same holds true for the A-band as compared to the I-band in heart muscle cells. A sequestration of K⁺ was measured also in the rough endoplasmic reticulum (RER) of hepatocytes. The Cl^? concentration in mitochondria both in heart muscle and liver cells has been measured far in excess of what might be expected from a Nernstian distribution. A coupled inward Cl^? transport in mitochondria must, therefore, be assumed.     

Evaluation of fracture planes and cell morphology in complementary fractures of cultured cells in the frozen-hydrated state by field-emission secondary electron microscopy: feasibility for ion localization and fluorescence imaging studies

We have employed field-emission secondary electron microscopy (FESEM) for morphological evaluation of freeze-fractured frozen-hydrated renal epithelial LLC-PK₁ cells prepared with our simple cryogenic sandwich-fracture method that does not require any high-vacuum freeze-fracture instrumentation (Chandra et al. (1986) J. Microsc. 144 , 15–37). The cells fractured on the substrate side of the sandwich were matched one-to-one with their corresponding complementary fractured faces on the other side of the sandwich. The FESEM analysis of the frozen-hydrated cells revealed three types of fracture: (i) apical membrane fracture that produces groups of cells together on the substrate fractured at the ectoplasmic face of the plasma membrane; (ii) basal membrane fracture that produces basal plasma membrane-halves on the substrate; and (iii) cross-fracture that passes randomly through the cells. The ectoplasmic face (E-face) and protoplasmic face (P-face) of the membrane were recognized based on the density of intramembranous particles. Feasibility of fractured cells was shown for intracellular ion localization with ion microscopy, and fluorescence imaging with laser scanning confocal microscopy. Ion microscopy imaging of freeze-dried cells fractured at the apical membrane revealed well-preserved intracellular ionic composition of even the most diffusible ions (total concentrations of K⁺, Na⁺ and Ca⁺). Structurally damaged cells revealed lower K⁺ and higher Na⁺ and Ca⁺ contents than in well-preserved cells. Frozen-freeze-dried cells also allowed imaging of fluorescently labelled mitochondria with a laser scanning confocal microscope. Since these cells are prepared without washing away the nutrient medium or using any chemical pretreatment to affect their native chemical and structural makeup, the characterization of fracture faces introduces ideal sample types for chemical and morphological studies with ion and electron microscopes and other techniques such as laser scanning confocal microscopy, atomic force microscopy and near-field scanning optical microscopy.     

Quasicrystalline Materials

SIMS matrix effects (mass interferences, sputter yield variations and practical ion yield variations) were evaluated in freeze-fractured, freeze-dried cultured cells at the ～0.5 μm spatial resolution of the Cameca IMS-3f ion microscope. Cell lines studied include normal rat kidney (NRK), 3T3 mouse fibroblast, L6 rat myoblast, chinese hamster ovary (CHO) and rat kangaroo kidney (PtK2) cells. High mass resolution studies indicated that the secondary ion signals of H^—, C^—, O^—, Na⁺, Mg⁺, CN^—, P^—, S^—, Cl^—, K⁺ and Ca⁺ were free from major mass interferences. However, a large mass interference was observed for nitrogen at mass 14. No significant sputtering yield difference between the nuclear and cytoplasmic compartments of the cells studied was observed. The subcellular distributions of the major (H, C, N and O) and minor (P, S, K, Cl, Na, Mg and Ca) matrix elements were found to be largely homogeneous with the exception of Ca, which was observed mainly in the cell cytoplasm. Practical ion yield variations were compared by three different approaches: (i) by the use of cells doped with known electrolyte concentrations, (ii) by quantitative ion implantation, and (iii) by analysis of the same cell with both electron probe and ion microscope. Each approach indicated an absence of significant practical ion yield differences between the nuclear and cytoplasmic regions of these specimens. These observations indicate that secondary ion signals in this type of sample are not significantly affected by local matrix effect variations. Hence, qualitative imaging of such specimens provides a true representation of subcellular elemental distribtions. These observations should allow the development of quantitative ion imaging methodologies and enhance the applicability of ion microscopy to biomedical problems.     

Morphological and ultrastructural characterization of ionoregulatory cells in the teleost oreochromis niloticus following salinity challenge combining complementary confocal scanning laser microscopy and transmission electron microscopy using a novel prefixation immunogold labeling technique

Aspects of ionoregulatory or mitochondria‐rich cell (MRC) differentiation and adaptation in Nile tilapia yolk‐sac larvae following transfer from freshwater to elevated salinities, that is, 12.5 and 20 ppt are described. Investigations using immunohistochemistry on whole‐mount Nile tilapia larvae using anti‐ Na⁺/K⁺‐ATPase as a primary antibody and Fluoronanogold? (Nanoprobes) as a secondary immunoprobe allowed fluorescent labeling with the high resolution of confocal scanning laser microscopy combined with the detection of immunolabeled target molecules at an ultrastructural level using transmission electron microscopy (TEM). It reports, for the first time, various developmental stages of MRCs within the epithelial layer of the tail of yolk‐sac larvae, corresponding to immature, developing, and mature MRCs, identifiable by their own characteristic ultrastructure and form. Following transfer to hyperosmotic salinities the density of immunogold particles and well as the intricacy of the tubular system appeared to increase. In addition, complementary confocal scanning laser microscopy allowed identification of immunopositive ramifying extensions that appeared to emanate from the basolateral portion of the cell that appeared to be correlated with the localization of subsurface tubular areas displaying immunogold labeled Na⁺/K⁺‐ATPase. This integrated approach describes a reliable and repeatable prefixation immunogold labeling technique allowing precise visualization of NaK within target cells combined with a 3D imaging that offers valuable insights into MRC dynamics at an ultrastructural level. Microsc. Res. Tech., 76:1016–1024, 2013. © 2013 Wiley Periodicals, Inc.     

Insight into 5-aminolevulinic acid-induced modulation of cellular antioxidant metabolism to confer salinity and drought tolerance in maize

The current study investigated the comparative oxidative damage in two maize seedlings induced by saline, drought, and combined stress and the ameliorative role of two different doses (20 and 80 µM) of 5-aminolevulinic acid (ALA) against the above-mentioned stresses. Hydroponically grown 10-day-old maize (Zea mays, var. BARI Hybrid Maize-7 (BHM-7) and BARI Hybrid Maize-9 (BHM-9)) seedlings were exposed to 12 dS/m of saline solution, 200 mM mannitol-induced drought stress alone and their combined stress for 7 days. Result revealed that individual stresses retard the plant growth to some degrees; however, their combined stress has more detrimental effects, which might be correlated with lipid peroxidation (MDA)-induced oxidative stress in seedlings, enhanced Na⁺ /K⁺ ratio, and augmented generation of superoxide (O₂^•− ) and hydrogen peroxide (H₂O₂). In contrast, exogenous ALA supplementation at 20 µM concentration markedly recovered from chlorosis and growth inhibition, substantially scavenged reactive oxygen species (ROS) and MDA by preserving ionhomeostasis and relaxing oxidative stress; also, by boosting catalase (CAT) and glutathione S-transferase (GST), and exclusively via depressing the activity of lipoxygenase (LOX) antioxidant enzyme. On the contrary, 80 µM ALA made things worse; nevertheless, higher activities shown by other antioxidant enzymes; like, superoxide dismutase (SOD), ascorbate peroxidase (APX), peroxidase (POD), and glutathione peroxidase (GPX), which were related to lessen the oxidative damage by highly produced O₂^•− and H₂O₂ under combined stress. Non-denaturing gel electrophoresis was done for further confirmation. However, ALA importantly increased the photosynthetic pigment contents in both genotypes irrespective of doses. Nevertheless, GST might have assisted the plants to escape from the herbicidal effect by detoxification. However, in the combined stress condition, high ALA concentration may have some positive role to play. Our findings also showed that BHM-9 performed better than BHM-7. Therefore, ALA at lower concentration was effective for single stress of saline and drought, while higher concentration can improve plant survival under combined stress.     

Effect of the polymeric cryoprotectant dextran on fluid secretion in the isolated rabbit pancreas

Physiological effects of the polymeric cryoprotectant dextran on an ion-transporting epithelium were investigated. In the isolated rabbit pancreas, dextran caused inhibition of fluid secretion and an increase of the concentrations of Na⁺, K⁺ and Cl^? in the secreted fluid. Dextran did not affect the basal or pancreozymin-stimulated enzyme secretion. These effects of dextran can partially be explained by the fact that it is osmotically active and does not permeate through the epithelium. The effect of dextran on water transport can be compensated by lowering the ion concentrations in the solvent of the cryoprotectant. It is concluded that in cryoprotected ion-transporting epithelia the absolute ion concentration values obtained by X-ray microanalysis of frozen-hydrated specimens may not be completely correct, but that valid conclusions about intracellular ion distribution may still be drawn.     

Confocal laser scanning microscopic analysis of ectopic sublingual gland‐like tissue inside the hamster submandibular gland

Based on its histochemical properties, the secretory portion of the hamster submandibular gland has been classified as seromucous cells. The presence of endogenous peroxidase (PO) reaction was shown in the nuclear envelope, cisternae of endoplasmic reticulum and Golgi apparatus. The 3,3′‐diaminobenzidene, tetrahydrochloride (DAB) method revealed bipartite secretory granules containing a PO‐positive dense core surrounded by a less dense halo in these cells. In the present investigation, serous and mucous‐like cells were found in resin‐embedded semi‐thin sections of the DAB‐reacted hamster submandibular gland. These sections were already on glass slides for routine light microscopic observations, therefore electron microscopic analysis could be unrealizable. We then used reflectance‐mode confocal laser scanning microscopy to visualize additional sites of PO activity as detected in these sections. Using this approach, we found mucous cells with PO activity‐negative secretory granules and seromucous cells with PO activity‐positive spot‐like secretory granules of the regular sublingual gland most frequently adjacent to the serous cells with typical electron‐dense secretory granules. These cells clearly differ from the seromucous cells with bipartite secretory granules and the granular duct cells with typical electron‐dense secretory granules of the hamster submandibular gland. Additionally, secretory endpieces of the ectopic sublingual gland‐like tissue empty into the duct of the hamster submandibular gland lobule. Thus, our findings suggest that a mass of sublingual gland tissue extends into the hamster submandibular gland during its development, and PO may be synthesized and secreted into the same duct. Microsc. Res. Tech. 76:1284–1291, 2013. © 2013 Wiley Periodicals, Inc.     

A new approach for high-pressure freezing of primary culture cells: the fine structure and stimulation-associated transformation of cultured rabbit gastric parietal cells

A newly designated procedure for high‐pressure freezing of primary culture cells provided excellent ultrastructure of rabbit gastric parietal cells. The isolated parietal cells were cultivated on Matrigel‐coated aluminium plates for conventional subsequential cryoimmobilization by high‐pressure freezing. The ultrastructure of different organelles (Golgi apparatus, mitochondria, multivesicular bodies, etc.) was well preserved compared to conventional chemical fixation. In detail, actin filaments were clearly shown within the microvilli and the subapical cytoplasm. Another striking finding on the cytoskeleton system is the abundance of microtubules among the tubulovesicles. Interestingly, some microtubules appeared to be associating with tubulovesicles. A large number of electron‐dense coated pits and vesicles were observed around the apical membrane vacuoles in cimetidine‐treated resting parietal cells, consistent with an active membrane uptake in the resting state. Immunogold labelling of H⁺/K⁺‐ATPase was seen on the tubulovesicular membranes. When stimulated with histamine, the cultured parietal cells undergo morphological transformation, resulting in great expansion of apical membrane vacuoles. Immunogold labelling of H⁺/K⁺‐ATPase was present not only on the microvilli of expanded apical plasma membrane vacuoles but also in the electron‐dense coated pits. The present findings provide a clue to vesicular membrane trafficking in cultured gastric parietal cells, and assure the utility of the new procedure for high‐pressure freezing of primary culture cells.     

Automated focusing in bright-field microscopy for tuberculosis detection

Pulsed‐laser atom‐probe tomography is used to compare the field‐evaporation mass spectrum and spatial distribution of molecular fragments from various poly(3‐alkylthiophene) films deposited on sharpened aluminium specimen carriers using two different deposition methods. Films deposited via a modified solution‐cast methodology yield small fragments with a uniform structural morphology whereas films deposited via an electrospray ionization methodology yield a wide range of fragments with a very non‐uniform structural morphology. The main field‐evaporated chemical species identified for both deposition types were, in order of typical relative abundance, C₂H₅⁺, CH₃⁺, C₂H₄⁺, followed by C₃H_7,8⁺/SC⁺ and SCH⁺. Thick electrospray depositions allowed investigation of the influence of laser‐pulse energy on the analysis. Evidence is presented supporting the presence of a critical laser‐pulse energy whereby changes in film morphology are signalled by the appearance of a new mass fragment at 190 Da.     

An X-ray microanalytical examination of precipitation methods for the ultrastructural localization of potassium in plant tissue: I. Cobaltinitrite

In vitro and in vivo studies were carried out to assess the feasibility of adapting the cobaltinitrite method for the ultrastructural localization of K⁺ in plant cells. The relatively low mobility of the cobaltinitrite ion and the solubility of the precipitation product resulted in the formation of only a few large deposits in each cell. The addition of Ag⁺ at the time of precipitation was found to increase the number of small deposits retained in sections. However, cobaltinitrite reagents frequently caused plasmolysis and poor cytoplasmic preservation. Precipitation during freeze-substitution was also attempted. It was concluded that precipitation of K⁺ using cobaltinitrite has only limited use in low magnification electron microscopy in conjunction with light microscopy.     

Electrical and electrochemical properties of the MEH–PPV and MEH–PPV doped calix[4]arene derivative layers for the detection of Cu and Na ions

A modified ITO electrode based on conducting polymer (MEH–PPV) matrix doped with the calix[4]arene molecules (β-ketoimine calix[4]arene) is deposited by spin coating. The carrier transport mechanism of the MEH–PPV and MEH–PPV:β-ketoiminecalix[4]arene films is investigated by using I–V characteristics measurements. The conduction mechanisms in these thin films are identified to be a space-charge-limited current (SCLC). The dielectric behavior of the MEH–PPV and the composite β-ketoimine calix[4]arene/MEH–PPV thin films are investigated by impedance spectroscopy (IS). The Cole–Cole plots are studied showing single semicircles and the solid interfaces are modeled by an equivalent circuit. The characteristics of chemical sensors based on MEH–PPV and MEH–PPV doped β-ketoimine calix[4]arene thin films for the detection of Cu²⁺ and Na⁺ ions are investigated by electrochemical impedance spectroscopy (EIS). The experimental impedance data of the modified electrodes are modeled by an equivalent circuit using the Z-View software. The inclusion of calixarene molecules into conducting polymer (MEH–PPV) exhibited considerable rise charge transfer resistance and improves sensing properties.     

Chronic alcohol consumption promotes alterations on salivary gland regeneration process

The aim of this study is to investigate the histological effect of alcohol ingestion on the regeneration of the submandibular gland (SMG) in rats. Twelve 60‐day‐old male Wistar rats were randomized into two experimental groups. Test group (TG) animals ingested 40° GL of alcohol for 45 days before surgery, being its concentration gradually increased 10° GL/week for 4 weeks to achieve the final concentration of 40° GL. The control group (CG) received water during the whole experimental period. One‐third of the left SMG lobe was removed. Three and seven days after, the whole gland was excised and analyzed. In the TG, the inflammatory process was pronounced when comparing the CG on day 3. The inverse aspect was observed on day 7, associated with an advanced parenchyma development. Changes in laminin expression and glycoproteins production were observed in the TG, causing advanced morphogenesis and delay in cytodifferentiation during the salivary gland regeneration, probably due to alcohol effects. Animals who received ethanol showed alterations in the pattern of glandular regeneration. Microsc. Res. Tech. 76:1125–1130, 2013. © 2013 Wiley Periodicals, Inc.     

Influence of fiber orientation on abrasive wear of unidirectionally reinforced carbon fiber–polyetherimide composites

A composite with continuous carbon fibers (CF) (80% by vol.) and high performance thermoplastic polyetherimide (PEI) matrix was developed and evaluated for various mechanical properties as a function of fiber orientation angle (0°, 30°, 45°, 60° and 90°). It was observed that Young's modulus, Poisson's ratio, toughness and % strain decreased with the increase of fiber orientation angle with respect to loading direction. In-plane shear modulus was highest for fibers with 45°. Overall, unidirectional (UD) CF reinforcement enhanced all strength properties of PEI significantly. Composites with fibers in 0° (parallel to loading direction) proved best in almost all the properties. Tribological evaluation in abrasive wear mode under different loads and fiber orientations indicated that coefficient of friction (μ) and specific wear rate (K₀) decreased with load, in general. Comparatively low specific wear rate (K₀), (in the order of 0.7 1×10^?9 m³/Nm) was observed for 0° fiber orientation, while fibers in 90° showed almost three times higher wear rate. Overall fiber reinforcement in 0° orientations proved beneficial from both strength and tribological performance point of view. SEM proved useful to correlate wear rate with surface topography.     

Minimizing photodecomposition of flavin adenine dinucleotide fluorescence by the use of pulsed LEDs

Dynamic alterations in flavin adenine dinucleotide (FAD) fluorescence permit insight into energy metabolism‐dependent changes of intramitochondrial redox potential. Monitoring FAD fluorescence in living tissue is impeded by photobleaching, restricting the length of microfluorimetric recordings. In addition, photodecomposition of these essential electron carriers negatively interferes with energy metabolism and viability of the biological specimen. Taking advantage of pulsed LED illumination, here we determined the optimal excitation settings giving the largest fluorescence yield with the lowest photobleaching and interference with metabolism in hippocampal brain slices. The effects of FAD bleaching on energy metabolism and viability were studied by monitoring tissue pO₂, field potentials and changes in extracellular potassium concentration ([K⁺]_o). Photobleaching with continuous illumination consisted of an initial exponential decrease followed by a nearly linear decay. The exponential decay was significantly decelerated with pulsed illumination. Pulse length of 5 ms was sufficient to reach a fluorescence output comparable to continuous illumination, whereas further increasing duration increased photobleaching. Similarly, photobleaching increased with shortening of the interpulse interval. Photobleaching was partially reversible indicating the existence of a transient nonfluorescent flavin derivative. Pulsed illumination decreased FAD photodecomposition, improved slice viability and reproducibility of stimulus‐induced FAD, field potential, [K⁺]_o and pO₂ changes as compared to continuous illumination.