scholarly journals Enhanced photon collection enables four dimensional fluorescence nanoscopy of living systems

2018 ◽  
Author(s):  
Luciano A. Masullo ◽  
Andreas Bodén ◽  
Francesca Pennacchietti ◽  
Giovanna Coceano ◽  
Michael Ratz ◽  
...  
Keyword(s):  
Fluorescent Proteins ◽  
Embryonic Stem ◽  
Photon Flux ◽  
Brain Cells ◽  
Optical Sectioning ◽  
Low Light ◽  
4D Imaging ◽  
Fluorescence Nanoscopy ◽  
Entire Cell ◽  
Fine Structures

The theoretically unlimited spatial resolution of fluorescence nanoscopy often comes at the expense of time, contrast and increased dose of energy for recording. Here, we developed MoNaLISA, for Molecular Nanoscale Live Imaging with Sectioning Ability, a nanoscope capable of imaging structures at a scale of 45–65 nm within the entire cell volume at low light intensities (W–kW/cm2). Our approach, based on reversibly switchable fluorescent proteins, features three distinctly modulated illumination patterns crafted and combined in a novel way to gain fluorescence ON-OFF switching cycles and image contrast. By maximizing the detected photon flux MoNaLISA enables prolonged (40–50 frames) and large (50 x 50 μm2) recordings at 0.3–1.3 Hz with enhanced optical sectioning ability. We demonstrated the general use of our approach by 4D imaging of organelles and fine structures in epithelial human cells, colonies of mouse embryonic stem cells, brain cells, and organotypic tissues.

scholarly journals Ecophysiological, morphological, and biochemical traits of free-living Diplosphaera chodatii (Trebouxiophyceae) reveal adaptation to harsh environmental conditions

PROTOPLASMA ◽  
2021 ◽  
Author(s):  
Cynthia Medwed ◽  
Andreas Holzinger ◽  
Stefanie Hofer ◽  
Anja Hartmann ◽  
Dirk Michalik ◽  
...  
Keyword(s):  
Environmental Conditions ◽  
Botanical Garden ◽  
Tree Bark ◽  
Photon Flux ◽  
Low Light ◽  
Transmission Electron ◽  
Light Requirements ◽  
Adaptive Mechanisms ◽  
Terrestrial Habitats ◽  
Excellent Preservation

AbstractSingle-celled green algae within the Trebouxiophyceae (Chlorophyta) are typical components of terrestrial habitats, which often exhibit harsh environmental conditions for these microorganisms. This study provides a detailed overview of the ecophysiological, biochemical, and ultrastructural traits of an alga living on tree bark. The alga was isolated from a cypress tree in the Botanical Garden of Innsbruck (Austria) and identified by morphology and molecular phylogeny as Diplosphaera chodatii. Transmission electron microscopy after high-pressure freezing (HPF) showed an excellent preservation of the ultrastructure. The cell wall was bilayered with a smooth inner layer and an outer layer of polysaccharides with a fuzzy hair-like appearance that could possibly act as cell-cell adhesion mechanism and hence as a structural precursor supporting biofilm formation together with the mucilage observed occasionally. The photosynthetic-irradiance curves of D. chodatii indicated low light requirements without photoinhibition at high photon flux densities (1580 μmol photons m−2 s−1) supported by growth rate measurements. D. chodatii showed a high desiccation tolerance, as 85% of its initial value was recovered after controlled desiccation at a relative humidity of ~10%. The alga contained the low molecular weight carbohydrates sucrose and sorbitol, which probably act as protective compounds against desiccation. In addition, a new but chemically not elucidated mycosporine-like amino acid was detected with a molecular mass of 332 g mol−1 and an absorption maximum of 324 nm. The presented data provide various traits which contribute to a better understanding of the adaptive mechanisms of D. chodatii to terrestrial habitats.

scholarly journals Measuring fast gene dynamics in single cells with time-lapse luminescence microscopy

2014 ◽  
Vol 25 (22) ◽  
pp. 3699-3708 ◽  
Author(s):  
Anyimilehidi Mazo-Vargas ◽  
Heungwon Park ◽  
Mert Aydin ◽  
Nicolas E. Buchler
Keyword(s):  
Gene Expression ◽  
Fluorescent Proteins ◽  
Single Cells ◽  
Time Lapse ◽  
Photon Flux ◽  
Luminescence Microscopy ◽  
Cell Cycle Genes ◽  
Light Excitation ◽  
Better Than

Time-lapse fluorescence microscopy is an important tool for measuring in vivo gene dynamics in single cells. However, fluorescent proteins are limited by slow chromophore maturation times and the cellular autofluorescence or phototoxicity that arises from light excitation. An alternative is luciferase, an enzyme that emits photons and is active upon folding. The photon flux per luciferase is significantly lower than that for fluorescent proteins. Thus time-lapse luminescence microscopy has been successfully used to track gene dynamics only in larger organisms and for slower processes, for which more total photons can be collected in one exposure. Here we tested green, yellow, and red beetle luciferases and optimized substrate conditions for in vivo luminescence. By combining time-lapse luminescence microscopy with a microfluidic device, we tracked the dynamics of cell cycle genes in single yeast with subminute exposure times over many generations. Our method was faster and in cells with much smaller volumes than previous work. Fluorescence of an optimized reporter (Venus) lagged luminescence by 15–20 min, which is consistent with its known rate of chromophore maturation in yeast. Our work demonstrates that luciferases are better than fluorescent proteins at faithfully tracking the underlying gene expression.

scholarly journals A simple Agrobacterium-mediated stable transformation technique for the hornwort model Anthoceros agrestis

2021 ◽  
Author(s):  
Eftychios Frangedakis ◽  
Manuel Waller ◽  
Tomoaki Nishiyama ◽  
Hirokazu Tsukaya ◽  
Xia Xu ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Genetic Manipulation ◽  
Fluorescent Proteins ◽  
Land Plant ◽  
Plant Evolution ◽  
Stable Transformation ◽  
35S Promoter ◽  
Low Light ◽  
Transformation Technique ◽  
High Transformation

We have developed a simple Agrobacterium-mediated method for the stable transformation of the hornwort Anthoceros agrestis, the fifth bryophyte species for which a genetic manipulation technique becomes available. High transformation efficiency was achieved by using thallus tissue grown under low-light conditions. We generated a total of 216 transgenic A. agrestis lines expressing the β-Glucuronidase (GUS), cyan, green, and yellow fluorescent proteins under the control of the CaMV 35S promoter and several endogenous promoters. Nuclear and plasma membrane localization with multiple color fluorescent proteins was also confirmed. The transformation technique described here should pave the way for detailed molecular and genetic studies of hornwort biology, providing much needed insight into the molecular mechanisms underlying symbiosis, carbon-concentrating mechanism, RNA editing, and land plant evolution in general.

Responses of Rainforest Understorey Plants to Excess Light during Sunflecks

1997 ◽  
Vol 24 (1) ◽  
pp. 17 ◽  
Author(s):  
Jenny R. Watling ◽  
Sharon A. Robinson ◽  
Ian E. Woodrow ◽  
C. Barry Osmond
Keyword(s):  
Tropical Rainforest ◽  
Photon Flux ◽  
Photochemical Quenching ◽  
Photon Flux Density ◽  
Leaf Angle ◽  
Low Light ◽  
Fourth Species ◽  
Excess Light ◽  
Understorey Plants ◽  
Non Photochemical Quenching

Responses of Alocasia macrorrhiza (L.) G. Don, Castanospora alphandii (F. Muell.) F. Muell. and Alpinia hylandii R. Smith, growing in a tropical rainforest understorey, to excess light during sunflecks were investigated using chlorophyll fluorescence techniques and by analysing xanthophyll cycle activity. A fourth species, the pioneerOmalanthus novo-guineensis (Warb.) Schum., growing in a small gap, was also studied. In all three understorey species there were large and rapid decreases in the proportion of open Photosystem II (PSII) centres, as indicated by qP, on illumination with saturating light and a concurrent increase in non-photochemical quenching. qP remained low (< 0.4) throughout the period of illumination (~15 min), although it did increase gradually, probably reflecting photosynthetic induction. Sustained declines (up to 120 min) in quantum yield, indicated by Fv/Fm, occurred in all three understorey species following exposure to saturating Photon flux density (PFD) during sunflecks. When ?PSII was monitored during sunflecks it was found to be very sensitive to changes in PFD, declining rapidly with even modest rises in the latter. There was rapid and continuing net conversion of violaxanthin (V) to antheraxanthin plus zeaxanthin (A+Z) on exposure of A. macrorrhiza and C. alphandii to saturating sunflecks. On returning to low light A. macrorrhiza retained its high levels of A+Z for up to 60 min, while C. alphandii rapidly converted back to V. O. novo- guineensis responded to high light by changing its leaf angle to reduce interception and showed no indication of photoinhibition during or after exposure.

scholarly journals Diverse Cell Types, Circuits, and Mechanisms for Color Vision in the Vertebrate Retina

2019 ◽  
Vol 99 (3) ◽  
pp. 1527-1573 ◽  
Author(s):  
Wallace B. Thoreson ◽  
Dennis M. Dacey
Keyword(s):  
Color Vision ◽  
Ganglion Cells ◽  
Color Perception ◽  
Visible Spectrum ◽  
Cell Types ◽  
Photon Flux ◽  
Low Light ◽  
Vertebrate Retina ◽  
Light Levels ◽  
Synaptic Interactions

Synaptic interactions to extract information about wavelength, and thus color, begin in the vertebrate retina with three classes of light-sensitive cells: rod photoreceptors at low light levels, multiple types of cone photoreceptors that vary in spectral sensitivity, and intrinsically photosensitive ganglion cells that contain the photopigment melanopsin. When isolated from its neighbors, a photoreceptor confounds photon flux with wavelength and so by itself provides no information about color. The retina has evolved elaborate color opponent circuitry for extracting wavelength information by comparing the activities of different photoreceptor types broadly tuned to different parts of the visible spectrum. We review studies concerning the circuit mechanisms mediating opponent interactions in a range of species, from tetrachromatic fish with diverse color opponent cell types to common dichromatic mammals where cone opponency is restricted to a subset of specialized circuits. Distinct among mammals, primates have reinvented trichromatic color vision using novel strategies to incorporate evolution of an additional photopigment gene into the foveal structure and circuitry that supports high-resolution vision. Color vision is absent at scotopic light levels when only rods are active, but rods interact with cone signals to influence color perception at mesopic light levels. Recent evidence suggests melanopsin-mediated signals, which have been identified as a substrate for setting circadian rhythms, may also influence color perception. We consider circuits that may mediate these interactions. While cone opponency is a relatively simple neural computation, it has been implemented in vertebrates by diverse neural mechanisms that are not yet fully understood.

Photoswitchable fluorescent proteins enable monochromatic multilabel imaging and dual color fluorescence nanoscopy

2008 ◽  
Vol 26 (9) ◽  
pp. 1035-1040 ◽  
Author(s):  
Martin Andresen ◽  
Andre C Stiel ◽  
Jonas Fölling ◽  
Dirk Wenzel ◽  
Andreas Schönle ◽  
...  
Keyword(s):  
Fluorescent Proteins ◽  
Dual Color ◽  
Fluorescence Nanoscopy

Invasion by Pittosporum undulatum of the Forests of Central Victoria. III. Effects of Temperature and Light on Growth and Drought Resistance

1982 ◽  
Vol 30 (3) ◽  
pp. 347 ◽  
Author(s):  
RM Gleadow ◽  
KS Rowan
Keyword(s):  
Leaf Area ◽  
Photon Flux ◽  
First Year ◽  
Low Light ◽  
Relative Growth ◽  
Relative Growth Rates ◽  
Drought Tolerant ◽  
Deep Shade ◽  
Net Assimilation ◽  
Shade Plants

The clumping of invading seedlings of Pittosporum undulatum Vent. around the butts of established trees is due partly to the site-dependent survival of seedlings. particularly during summer. P. undulatum seedlings were very drought-tolerant when grown at moderately low temperatures (21.4°C day/17.8°C night compared with 27.4°C day/23.9°C night) and in deep shade. Plants droughted at 5°C higher temperatures and higher photon flux densities stopped transpiring and wilted 10-13 days earlier than those droughted under cooler, densely shaded conditions. Well watered seedlings had higher relative growth rates and net assimilation rates when grown under higher temperatures and photon flux densities. Control seedlings were more leafy when grown under low light. as reflected by the specific leaf area, leaf area ratio and root/shoot ratios. The adverse effects of higher photon flux densities and temperatures on the drought tolerance of P. undulatum seedlings support the hypothesis that survlval of invading seedlings in their first year is dependent on the microclimate.

scholarly journals Response of CO 2 -starved diatom Phaeodactylum tricornutum to light intensity transition

2017 ◽  
Vol 372 (1728) ◽  
pp. 20160396 ◽  
Author(s):  
Parisa Heydarizadeh ◽  
Wafâa Boureba ◽  
Morteza Zahedi ◽  
Bing Huang ◽  
Brigitte Moreau ◽  
...  
Keyword(s):  
Flux Density ◽  
Carbon Metabolism ◽  
Phaeodactylum Tricornutum ◽  
Shikimate Pathway ◽  
Aromatic Amino Acids ◽  
Molecular Data ◽  
High Light ◽  
Photon Flux ◽  
Photon Flux Density ◽  
Low Light

In this study, we investigated the responses of Phaeodactylum tricornutum cells acclimated to 300 µmol m −2 s −1 photon flux density to an increase (1000 µmol m −2 s −1 ) or decrease (30 µmol m −2 s −1 ) in photon flux densities. The light shift occurred abruptly after 5 days of growth and the acclimation to new conditions was followed during the next 6 days at the physiological and molecular levels. The molecular data reflect a rearrangement of carbon metabolism towards the production of phosphoenolpyruvic acid (PEP) and/or pyruvate. These intermediates were used differently by the cell as a function of the photon flux density: under low light, photosynthesis was depressed while respiration was increased. Under high light, lipids and proteins accumulated. Of great interest, under high light, the genes coding for the synthesis of aromatic amino acids and phenolic compounds were upregulated suggesting that the shikimate pathway was activated. This article is part of the themed issue ‘The peculiar carbon metabolism in diatoms’.

scholarly journals Photochemical Acclimation of Three Contrasting Species to Different Light Levels: Implications for Optimizing Supplemental Lighting

2017 ◽  
Vol 142 (5) ◽  
pp. 346-354 ◽  
Author(s):  
Shuyang Zhen ◽  
Marc W. van Iersel
Keyword(s):  
Electron Transport ◽  
Light Intensity ◽  
Photochemical Efficiency ◽  
Light Response ◽  
High Light ◽  
Photon Flux ◽  
Ambient Light ◽  
Response Curves ◽  
Low Light ◽  
Supplemental Lighting

Photosynthetic responses to light are dependent on light intensity, vary among species, and can be affected by acclimation to different light environments (e.g., light intensity, spectrum, and photoperiod). Understanding how these factors affect photochemistry is important for improving supplemental lighting efficiency in controlled-environment agriculture. We used chlorophyll fluorescence to determine photochemical light response curves of three horticultural crops with contrasting light requirements [sweetpotato (Ipomea batatas), lettuce (Lactuca sativa), and pothos (Epipremnum aureum)]. We also quantified how these responses were affected by acclimation to three shading treatments-full sun, 44% shade, and 75% shade. The quantum yield of photosystem II (ΦPSII), a measure of photochemical efficiency, decreased exponentially with increasing photosynthetic photon flux (PPF) in all three species. By contrast, linear electron transport rate (ETR) increased asymptotically with increasing PPF. Within each shading level, the high-light-adapted species sweetpotato used high light more efficiently for electron transport than light-intermediate lettuce and shade-tolerant pothos. Within a species, plants acclimated to high light (full sun) tended to have higher ΦPSII and ETR than those acclimated to low light (44% or 75% shade). Nonphotochemical quenching (NPQ) (an indicator of the amount of absorbed light energy that is dissipated as heat) was upregulated with increasing PPF; faster upregulation was observed in pothos as well as in plants grown under 75% shade. Our results have implications for supplemental lighting: supplemental light is used more efficiently and results in a greater increase in ETR when provided at low ambient PPF. In addition, high-light-adapted crops and crops grown under relatively high ambient light can use supplemental light more efficiently than low-light-adapted crops or those grown under low ambient light.

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