Microscope laser light scattering spectroscopy of single biological cells

A microscope laser light scattering setup was developed, allowing us to do intensity autocorrelation spectroscopy on the light scattered from a volume as small as (2 μm)³. This non-invasive technique makes cytoplasmic studies possible inside single live biological cells. The effect of osmotic swelling and shrinking on the diffusion coefficient of hemoglobin inside intact red blood cells is shown as an illustrative example of the applicability and sensitivity of this new experimental method.     

Luminol as the light source for in situ photodynamic therapy

Presently, the light sources used in photodynamic therapy are high intensity lasers or light emitting diodes, making it unsuitable for large-volume tumors and those located deep inside the body. To overcome this limitation, we propose an in situ light source to excite the photosensitizer to generate toxic singlet oxygen and kill tumor cells directly. In this research, luminol served as the in situ light source in 5-aminolevulinic acid-mediated photodynamic treatment of Caco-2 cell cultures. 72 h after luminol excitation the viability of the treated cells significantly decreased compared to the control cells in assays including cell viability, cytotoxicity, flow cytometry and fluorescence confocal microscopy. According to the results, we suggested luminol could be used as an in situ light source for 5-aminolevulinic acid-mediated photodynamic therapy. This method would have great potential to extend the application of photodynamic therapy to tumors located deep inside the body.     

Simultaneous visualization of two antigens in the same tissue section by combining immunoperoxidase with immunofluorescence techniques.

A simple method was developed whereby immunoperoxidase and immunofluorescence techniques were applied in consecutive steps to demonstrate the presence of two antigens in the same tissue section. This method was applied in three model, two antigens were shown: a) each (gastrin and pepsinogen II) inside one of two different cell types (gastrin (G) and antral peptic cells), b) each (kappa or gamma light chains) inside different cells of the same type (plasma cells); also, both (kamma and gamma light chains) inside the same cell (Reed-Sternberg cell), and c) both (pepsinogen I and II) inside the same cell (chief cell of oxyntic glands). The results could be viewed and photographed either simultaneously, when the antigens were in different cells, or sequentially, when the antigens were in the same cells.     

Buder revisited: cell and organ polarity during phototropism

The induction of a radial polarity by environmental stimuli was studied at the cellular and organ levels, with phototropism chosen as a model. The light gradient acting on the whole coleoptile was opposed to the light direction acting upon individual cells in the classical Buder experiment, irradiating from the inside out. Alternatively, the stimulus was administered to the coleoptile tip with a microbeam-irradiation device. Tropistic curvature was assayed as a marker for the response of the whole organ, whereas cell elongation and the orientation of cortical microtubules were taken as markers for the responses of individual cells. Upon tip irradiation, signals much faster than basipetal auxin transport migrate towards the base. The data are discussed in terms of an organ polarity that is the primary result of the asymmetric light signal and affects, in a second step, an endogenous radial polarity of epidermal cells.     

A tunable fluorescent timer method for imaging spatial‐temporal protein dynamics using light‐driven photoconvertible protein

Cellular function is largely determined by protein behaviors occurring in both space and time. While regular fluorescent proteins can only report spatial locations of the target inside cells, fluorescent timers have emerged as an invaluable tool for revealing coupled spatial‐temporal protein dynamics. Existing fluorescent timers are all based on chemical maturation. Herein we propose a light‐driven timer concept that could report relative protein ages at specific sub‐cellular locations, by weakly but chronically illuminating photoconvertible fluorescent proteins inside cells. This new method exploits light, instead of oxygen, as the driving force. Therefore its timing speed is optically tunable by adjusting the photoconverting laser intensity. We characterized this light‐driven timer method both in vitro and in vivo and applied it to image spatiotemporal distributions of several proteins with different lifetimes. This novel timer method thus offers a flexible “ruler” for studying temporal hierarchy of spatially ordered processes with exquisite spatial‐temporal resolution. (© 2015 WILEY‐VCH Verlag GmbH &Co. KGaA, Weinheim)     

Detergent treatment of Dictyostelium discoideum cells allows examination of internal cell type-specific antigens by flow cytometry

Monoclonal antibodies are used extensively in flow cytometry to identify subpopulations of cells differing in surface antigens. Conventional studies on living cells do not allow analysis of internal antigens, because antibody molecules do not pass through an intact plasma membrane. It is important for developmental studies on Dictyostelium discoideum that not only surface but also internal antigens be analysed. Here techniques are reported that make possible such studies by permeabilising cells with mild detergent treatments using digitonin. Flow cytometer profiles of unfixed cells show that antigens recognised by two monoclonal antibodies, MUD102 and MUD3, are found inside subpopulations of cells in the D. discoideum slug. Double-labelling experiments were carried out to demonstrate that the antigens recognised by these antibodies are present inside prespore but not prestalk cells. The detergent treatment leads to loss of forward-angle light scatter, but 90 degrees light scatter of cells is not greatly affected. While fixed cells sometimes gave satisfactory results, internal labelling did not reliably demonstrate the two subpopulations observed with unfixed cells.     

Use of green fluorescent protein to visualize the early events of symbiosis between Rhizobium meliloti and alfalfa (Medicago sativa).

A gene encoding a variant of green fluorescent protein (GFP) of Aequorea victoria was put under the control of a promoter which is constitutive in Rhizobium meliloti. The heterologous GFP gene was expressed at high levels during all stages of symbiosis, allowing R. meliloti cells to be visualized as they grew in the rhizosphere, on the root surface, and inside infection threads. In addition, nodules that were infected with bacteria which were synthesizing GFP fluoresced when illuminated with blue light. GFP-tagged bacteria could be seen inside infection threads, providing the opportunity to measure the growth rate and determine the patterns of growth of R. meliloti residing inside its host plant.     

Enzyme-assisted photosensitization with rose Bengal acetate induces structural and functional alteration of mitochondria in HeLa cells

Rose Bengal acetate (RB-Ac) can be used as a fluorogenic substrate for photosensitization of cells both in vivo and in vitro: once inside the cells, RB-Ac is converted into photoactive rose Bengal (RB) molecules which redistribute dynamically in the cytoplasm and, upon irradiation by visible green light, can damage organelles such as the endoplasmic reticulum, the Golgi apparatus, and the cytoskeleton. Recently, evidence has been provided that mitochondria may also be affected. The aims of the present study were to describe RB-induced photodamage of mitochondria in single HeLa cells and to define, on a quantitative basis, the effects of photosensitization on their morphofunctional features. HeLa cell cultures were exposed to 10⁻⁵ M RB-Ac for 60 min and then irradiated with a light emitting diode at 530 nm (total light dose, 1.6 J/cm²). After irradiation, the cells were transferred to a drug-free complete medium and allowed to grow for 24–72 h. Using conventional and confocal fluorescence microscopy, transmission electron microscopy, and flow cytometry, we demonstrate that, in photosensitized cells, mitochondria undergo structural and functional alterations which can lead cells to apoptosis. Interestingly, in our system some cells were able to survive 72 h post-treatment and to recover, exhibiting the same mitochondrial structure, distribution and inner membrane potential as those in untreated controls. Taking into account that the photoactive molecules redistribute dynamically inside the cell upon RB-Ac administration, it may be hypothesized that cells can be differently affected by irradiation, depending on the relative amount and organelle location of the photosensitizer.     

The influence of epidermal windows on the light environment within the leaves of six succulents

An omni-directional fibre optic microprobe was used to measure the quantity and quality of light within the leaves of six succulents having epidermal windows, three species having a subterranean growth habit (Haworthia truncata, Lithops olivacea, and Opthalmophyllum longum) and three growing above ground (Peperomia dolabriformis, P. graveolens, and the sprawling vine Senecio rowleyanus). Although light levels at most locations inside the leaves of all species were high, near those incident on the window surfaces, light levels inside the leaves of the two species of Peperomia often greatly exceeded incident light levels, indicating considerable light scattering and focusing by the leaf tissue. The spectral quality of light inside the leaves of all taxa reflected the absorption properties of chlorophyll, with most of the photons in the green wavelengths. Light quality and quantity inside the leaves did not correlate with the growth habit of the plants, the size of the window (as a proportion of the total leaf area), or location inside the leaf, although light levels generally declined and wavelengths increased deeper in the leaves. Application of reflective tape to the windows reduced internal light levels in L. olivacea and S. rowleyanus, although reductions were not always statistically significant. Although light levels throughout the leaves of P. graveolens were substantially and significantly reduced as a result of the application of reflective tape to its windows, the light levels even at the basal chlorenchyma on the abaxial side of the leaf remained high. In all species investigated, the levels of near-infrared radiation inside the leaves were surprisingly high, yet also declined deeper inside the succulent leaves. This near-infrared radiation may add to the heat load of these plants. Furthermore, application of reflective tape to the windows also reduced the amount of near-infrared radiation inside the leaves of the three succulents examined. These results led to a novel, testable hypothesis that may help to explain previous findings that application of reflective tape to the windows of the leaves of these succulents did not effect a reduction in photosynthetic activity.     

A light distribution model for an internally radiating photobioreactor

Analysis of light energy distribution in culture is important for maximizing the growth efficiency of photosynthetic cells and the productivity of a photobioreactor. To characterize the irradiance conditions in a photobioreactor, we developed a light distribution model for a single-radiator system and then extended the model to multiple radiators using the concept of parallel translation. Mathematical expressions for the local light intensity and the average light intensity were derived for a cylindrical photobioreactor with multiple internal radiators. The proposed model was used to predict the irradiance levels inside an internally radiating photobioreactor using Synechococcus sp. PCC 6301 as a model photosynthetic microorganism. The effects of cell density and radiator number were interpreted through photographic and model simulation studies. The predicted light intensity values were found to be very close to those obtained experimentally, which suggests that the proposed model is capable of accurately interpreting the local light energy profiles inside the photobioreactor system. Due to the simplicity and flexibility of the proposed model, it was also possible to predict the light conditions in other complex photobioreactors, including optical-fiber and pond-type photobioreactors.     

Elastic and inelastic light scattering in flow cytometry

A review of the fundamental aspects of elastic light scattering suggests that information about the shape and internal structure may best be obtained from signals measured in the backscattering directions. Size information can be most readily extracted from forward scattering signals. Spectral analysis of scattered signals with incident white light is a subject that merits further study. Signals from cells that have been stained with fluorescent dyes are proportional to dye content in the usual flow-cytometric configuration except in the case of dense, strongly anisometric structures such as sperm cells. The recent discovery that Raman signals from molecules adsorbed on small silver particles are strongly enhanced suggests the possibility of utilizing this effect for identification of molecular species inside biological cells.     

Death Mechanism of Breast Adenocarcinoma Cells Caused by BRET-Induced Cytotoxicity of miniSOG Depends on the Intracellular Localization of the NanoLuc–miniSOG Fusion Protein

Photodynamic therapy (PDT) is widely used in clinical practice to influence neoplasms in the presence of a photosensitizer, oxygen, and light source. The main problem of PDT of deep tumors is the problem of delivering excitation light (without lost of its intensity) inside the body. An alternative to the external light sources can be the internal light sources based on luciferase–substrate bioluminescent systems. In our work, we used the NanoLuc–furimazine system as an internal light source. This system can be successfully used to excite the protein photosensitizer miniSOG and to induce the phototoxicity of this flavoprotein in cancer cells during bioluminescent resonance energy transfer (BRET). It was shown that the mechanism of cell death caused by BRET-induced phototoxicity of mimiSOG in the presence of furimazine depends on the intracellular localization of the NanoLuc–miniSOG fusion protein: BRET-mediated activation of miniSOG in mitochondrial localization causes apoptosis, while the membrane localization of PS causes necrosis of cancer cells.     

魔芋球茎贮藏淀粉和甘露聚糖粒的形态观察

在光学显微镜和透射电镜下观察了魔芋（Ａｍｏｒｐｈｏｐｈａｌｕｓｃｏｎｊａｃ）球茎中甘露聚糖粒和淀粉粒的形态。两种贮藏多糖分别位于不同的细胞中。淀粉粒在造粉体内发育,以复粒存在,用魔芋球茎仔茎茎尖为材料观察显示,淀粉粒的形成早于甘露聚糖颗粒的形成。甘露聚糖粒形态多数近随圆形,一些甘露聚糖颗粒内包含了针晶体,但多数的甘露聚糖粒内部不包含针晶体,由纯净的甘露聚糖构成。     

The effect of temperature on the turnover of taste bud cells in catfish.

Renewal of taste bud cells on the barbels of channel catfish was studied. Groups of catfish, held in and acclimitized to 14 degrees C, 18 degrees C, 22 degrees C and 30 degrees C dechlorinated tap water were injected with [3H]thymidine (3.0 muCi/g body weight intraperitoneally). Barbels were sampled at various times after injection and prepared for light microscope autoradiography. Results show that epithelial cells surrounding the taste buds divide and some of their daughter cells migrate into the taste buds. The time at which 50% of the labelled cells have degenerated is taken as the average turnover time or average life span of the taste bud cells. The average life span as well as the time spent inside the taste buds is highly temperature-dependent. At 14 degrees C, 18 degrees C, 22 degrees C and 30 degrees C the average life span is on the order of 40, 30, 15 and 12 days respectively. Further studies indicate that both light and dark staining cells of the taste bud were labelled.     

Ultrastructural changes of the microvillar cytoskeleton in the photoreceptor of the crayfish Orconectes limosus related to different adaptation conditions

In the retina of crayfish microvilli of seven of the eight photoreceptor cells build highly organized structures, the rhabdoms. Cytoskeletal elements inside the microvilli were investigated in conventional and slightly extracted electron microscopical preparations. In conventional preparations the ultrastructure of these cytoskeletal elements depended on the adaptational state of the animal. They appeared as central filament-like structures inside each microvillus when dark-adapted retinae were prepared and fixed at night in the absence of calcium. Changes of these conditions (light, daytime, or calcium concentration) impaired the detectability of these central filaments; in light-adapted eyes prepared at midday they were rarely seen. Nevertheless, single microvillar filaments were present in light-adapted retinae after mild cell permeabilization with the saponin beta-escin. They appeared as a regular structure in each microvillus, often attached to the membrane. Their fine structure was consistent with the ultrastructure of single actin filaments as indicated by fast-Fourier-analysis and further supported by the presence of anti-actin immunoreactivity in electron microscopical and immunocytochemical preparations. These results indicate that microvillar filaments are not necessarily destroyed by light as previously described; we suggest that their appearance inside the microvillus might be altered by the properties of associated, maybe sidearm-like proteins.     

The detection of albumin intraperitoneally administered to rabbits in the subclavian lymph node]

With light and electron microscopy, the localization of human albumin labeled with colloidal gold is described in the subclavia lymph nodes of rabbits following an intraperitoneal injection of this labeled albumin. Most of the particles were found in the reticular cells of the sinus, and some particles were identified in the sinus macrophages. No particles were found inside lymph node follicules within 1 hour after injection. All stages of internalization of foreign protein inside lymph node cells were demonstrated.     

Culture and nitrite uptake in immobilized Scenedesmus obliquus

Summary The green alga Scenedesmus obliquus was immobilized in Ca-alginate beads. The cell growth after immobilization was studied by cell counting. The nitrite uptake was not affected by immobilization, except that a longer lag phase was observed in immobilized cells than in free ones. That result could be due to a barrier effect of the matrix against nitrite diffusion inside the beads. The treatment of cells by glycerol prior to their immobilization in a batch reactor induced an increase of nitrite uptake by the cells. This effect disappeared after a few runs. The glycerol effect on specific rates seemed also to decrease when the number of immobilized cells increased. This decrease can be related to the decrease of light efficiency as well as substrate accessibility when a high cell concentration was used. Several alternating runs of Tris-HCl buffer containing nitrite growth medium depleted in combined nitrogen were tested. Cellular growth occurred inside the beads up to a maximum followed by a decrease of cell number in the beads.     

Entry and survival of pathogenic mycobacteria in macrophages

Pathogenic mycobacteria, including Mycobacterium tuberculosis, are phagocytosed by macrophages but manage to survive within the mycobacterial phagosome. Recent work has shed some more light on the mechanisms of mycobacterial entry and survival inside macrophages. Two host cell components, the steroid cholesterol and a phagosomal coat protein termed TACO were found to play crucial roles in the establishment of an intracellular infection. This review describes how these findings may help to understand the circumvention of the normal trafficking routes inside host cells by mycobacteria.     

Dwarf male of Symbion pandora (cycliophora)

This study clarifies the identity and development of the male in the life cycle of Symbion pandora. The male is not produced directly by the feeding stage, as previously thought, but arises as a distinct individual from budding cells inside an intermediate stage named the Prometheus larva. The morphology and the development of the two distinct stages are described with light and electron microscopy. Furthermore, the following terminology is suggested to clearly distinguish between the different individuals: 1) the Prometheus larva, which is the free-swimming individual being produced inside the feeding stage; 2) the attached Prometheus larva on the feeding stage, which mostly degenerates following settlement, except for the internal budding cells; and 3) the dwarf male, which is the ciliated, sexually mature stage. The budding cells inside the attached Prometheus larva usually develop two internal dwarf males. Each dwarf male is heavily ciliated and has a well-developed nervous system with a relatively large brain, numerous gland and muscle cells, testis with bundles of sperm, and one penial structure. The male lacks a gut, as in the other free stages in the life cycle of Symbion pandora. This study also indicates that the dwarf male is freed from the attached Prometheus larva. Copulation, which has not been observed yet, probably takes place between a free-swimming male and the female, either while the female is released or afterwards.     

INCOMPLETENESS OF THE COCCOSPHERE AS A POSSIBLE STIMULUS FOR COCCOLITH FORMATION IN PLEUROCHRYSIS CARTERAE (PRYMNESIOPHYCEAE)1

Pleurochrysis carterae is a marine biflagellate that produces calcified structures called coccoliths. The coccoliths are formed inside the cells and released from the latter after formation. The light dependence of calcium incorporation in this species was studied using ⁴⁵Ca as a tracer. Cells exposed to a repeating cycle of 16 h of light and 8 h of darkness incorporated calcium in extracellular coccoliths at a more or less constant rate throughout a cycle. The cells divided during the dark periods with a concomitant decrease in size. Their size increased during the light periods. Coccolith formation in cells incubated in continuous darkness was greatly reduced and finally ceased. These cells did not divide and did not increase in size. Removal of extracellular coccoliths prior to the calcium incorporation experiments stimulated coccolith formation both in dark-incubated cells and in cells exposed to a repeating light-dark cycle. Cells in the stationary phase of growth ceased producing coccoliths. Calcification could be induced in these cells by removal of the extracellular coccoliths. Based on these findings we suggest that cells of Pleurochrysis carterae tend to produce a complete cover of coccoliths and that the available cell surface is a factor controlling coccolith formation.