Development of a non-invasive method to detect pericellular spatial oxygen gradients using FLIM

PhDExtracellular oxygen concentrations affect cellular metabolism and influence tissue function. Detection methods for these extracellular oxygen concentrations currently have poor spatial resolution and are frequently invasive. Fluorescence Lifetime Imaging Microscopy (FLIM) offers a non-invasive method for quantifying local oxygen concentrations. However, existing FLIM methods also show limited spatial resolution >1 μm and low time-resolved accuracy and precision, due to widefield time-gate. This study describes a new optimised approach using FLIM to quantity extracellular oxygen concentration with high accuracy (±7 μmol/kg) and spatial resolution ( ≅ 0.3 μm). An oxygen sensitive fluorescent dye, tris(2,2′-bipyridyl)ruthenium(II) chloride hexahydrate [Ru(bipy)3]+2, was excited with a multi-photon laser and fluorescence lifetime was measured using time-correlated single photon counting (TCSPC). The system was fully calibrated with optimised techniques developed for avoiding artefacts associated with photon pile-up and phototoxicity, whilst maximising spatial and temporal resolution. An extended imaging protocol (1800 sec) showed no phototoxic effects on cells at dye concentrations of <0.4 mM. Extracellular spatial oxygen gradients were identified around isolated chondrocytes, seeded in three-dimensional agarose gel. The technique was validated by regulating oxygen cellular consumption and thus confirming that the oxygen gradient was governed by cellular consumption. The technique identified a subpopulation of cells exhibiting statistically significant spatial oxygen gradients at the cell perihery. The subpopulation was shown to be significantly larger in cell diameter correlating with what that expected from chondrocytes in the deep zone. This technique provides an exciting opportunity to non-invasively quantify pericellular spatial oxygen gradients from within three-dimensional cellular constructs without prior manipulation of the cells. Thus by examining cellular metabolisms it will advance our understanding of the optimal cellular environment for tissue engineering and regenerative medicine

Planar Airy beam light-sheet for two-photon microscopy

We demonstrate the first planar Airy light-sheet microscope. Fluorescence light-sheet microscopy has become the method of choice to study large biological samples with cellular or sub-cellular resolution. The propagation-invariant Airy beam enables a ten-fold increase in field-of-view with single-photon excitation; however, the characteristic asymmetry of the light-sheet limits its potential for multi-photon excitation. Here we show how a planar light-sheet can be formed from the curved propagation-invariant Airy beam. The resulting symmetric light sheet excites two-photon fluorescence uniformly across an extended field-of-view without the need for deconvolution. We demonstrate the method for rapid two-photon imaging of large volumes of neuronal tissue.Comment: 7 pages, 4 figure

Application of Airy beam light sheet microscopy to examine early neurodevelopmental structures in 3D hiPSC-derived human cortical spheroids.

BACKGROUND: The inability to observe relevant biological processes in vivo significantly restricts human neurodevelopmental research. Advances in appropriate in vitro model systems, including patient-specific human brain organoids and human cortical spheroids (hCSs), offer a pragmatic solution to this issue. In particular, hCSs are an accessible method for generating homogenous organoids of dorsal telencephalic fate, which recapitulate key aspects of human corticogenesis, including the formation of neural rosettes-in vitro correlates of the neural tube. These neurogenic niches give rise to neural progenitors that subsequently differentiate into neurons. Studies differentiating induced pluripotent stem cells (hiPSCs) in 2D have linked atypical formation of neural rosettes with neurodevelopmental disorders such as autism spectrum conditions. Thus far, however, conventional methods of tissue preparation in this field limit the ability to image these structures in three-dimensions within intact hCS or other 3D preparations. To overcome this limitation, we have sought to optimise a methodological approach to process hCSs to maximise the utility of a novel Airy-beam light sheet microscope (ALSM) to acquire high resolution volumetric images of internal structures within hCS representative of early developmental time points. RESULTS: Conventional approaches to imaging hCS by confocal microscopy were limited in their ability to image effectively into intact spheroids. Conversely, volumetric acquisition by ALSM offered superior imaging through intact, non-clarified, in vitro tissues, in both speed and resolution when compared to conventional confocal imaging systems. Furthermore, optimised immunohistochemistry and optical clearing of hCSs afforded improved imaging at depth. This permitted visualization of the morphology of the inner lumen of neural rosettes. CONCLUSION: We present an optimized methodology that takes advantage of an ALSM system that can rapidly image intact 3D brain organoids at high resolution while retaining a large field of view. This imaging modality can be applied to both non-cleared and cleared in vitro human brain spheroids derived from hiPSCs for precise examination of their internal 3D structures. This process represents a rapid, highly efficient method to examine and quantify in 3D the formation of key structures required for the coordination of neurodevelopmental processes in both health and disease states. We posit that this approach would facilitate investigation of human neurodevelopmental processes in vitro

Super-Resolution Imaging Strategies for Cell Biologists Using a Spinning Disk Microscope

In this study we use a spinning disk confocal microscope (SD) to generate super-resolution images of multiple cellular features from any plane in the cell. We obtain super-resolution images by using stochastic intensity fluctuations of biological probes, combining Photoactivation Light-Microscopy (PALM)/Stochastic Optical Reconstruction Microscopy (STORM) methodologies. We compared different image analysis algorithms for processing super-resolution data to identify the most suitable for analysis of particular cell structures. SOFI was chosen for X and Y and was able to achieve a resolution of ca. 80 nm; however higher resolution was possible >30 nm, dependant on the super-resolution image analysis algorithm used. Our method uses low laser power and fluorescent probes which are available either commercially or through the scientific community, and therefore it is gentle enough for biological imaging. Through comparative studies with structured illumination microscopy (SIM) and widefield epifluorescence imaging we identified that our methodology was advantageous for imaging cellular structures which are not immediately at the cell-substrate interface, which include the nuclear architecture and mitochondria. We have shown that it was possible to obtain two coloured images, which highlights the potential this technique has for high-content screening, imaging of multiple epitopes and live cell imaging

Cartographie de la fluidité des membranes de spores de B. subtilis par microscopie de fluorescence résolue en temps

International audienceL’état de la membrane plasmique des cellules est un élément essentiel pour connaitre la conditionphysiologique des cellules. Sur des microorganismes, cette connaissance permet de mesurer l’impact d’uneperturbation sur la structure cellulaire et sur sa survie ultérieure. La fluidité membranaire résulte tout à lafois de la composition en phospholipides, de la présence et du rôle de certaines molécules comme lesstérols, les protéines transmembranaires mais aussi des conditions thermodynamiques et physico-chimiquesextérieures (P, T, aw). La bactérie Bacillus subtilis est capable dans des conditions défavorables de passerd’un état actif végétatif à l’état de spore, état de dormance accompagné d’un enkystement cellulaire. Danscet état, la structure unique de la spore comporte deux membranes phospholipidiques. La membraneinterne, la plus importante, présente une faible perméabilité sans modifications fondamentales de sacomposition (Griffith and Setlow, 2009). Elle a de plus un rôle essentiel dans l’extrême résistance de la spore,notamment aux attaques chimiques. Afin d’étudier cette transformation ainsi que la résistance de lamembrane de la spore aux différentes perturbations, nous avons développé un nouveau type de marquageafin de suivre la fluidité membranaire dans la cellule et dans la spore. Ce développement a été rendunécessaire car l’imperméabilité de la spore et la présence de deux membranes rendait difficile d’autresapproches. Ce marquage utilise un rotor moléculaire apolaire le Bodipy C12 (Kuimova, 2012). L’utilisation del’imagerie par temps de vie de fluorescence (FLIM) a permis de mesurer directement la microviscosité dumilieu qui environne la sonde ainsi que de différentier le signal venant de chaque membrane (Loison et al.2013). Ce marquage a permis de suivre l’état de la membrane lors de la germination ou lors de perturbationsenvironnementales (éthanol, température). Il devient ainsi possible de suivre l’état membranaire de la sporependant la perturbation. On peut ainsi espérer mieux comprendre comment cette membrane permet à laspore de résister à des conditions extrêmes et comment elle peut être altérée de façon irréversible ou non,pour certaines perturbations

Direct investigation of viscosity of an atypical inner membrane of Bacillus spores:a molecular rotor/FLIM study

AbstractWe utilize the fluorescent molecular rotor Bodipy-C12 to investigate the viscoelastic properties of hydrophobic layers of bacterial spores Bacillus subtilis. The molecular rotor shows a marked increase in fluorescence lifetime, from 0.3 to 4ns, upon viscosity increase from 1 to 1500cP and can be incorporated into the hydrophobic layers within the spores from dormant state through to germination. We use fluorescence lifetime imaging microscopy to visualize the viscosity inside different compartments of the bacterial spore in order to investigate the inner membrane and relate its compaction to the extreme resistance observed during exposure of spores to toxic chemicals. We demonstrate that the bacterial spores possess an inner membrane that is characterized by a very high viscosity, exceeding 1000cP, where the lipid bilayer is likely in a gel state. We also show that this membrane evolves during germination to reach a viscosity value close to that of a vegetative cell membrane, ca. 600cP. The present study demonstrates quantitative imaging of the microscopic viscosity in hydrophobic layers of bacterial spores Bacillus subtilis and shows the potential for further investigation of spore membranes under environmental stress