Resources
Brochures
Books
With contributions from renowned experts in the fields of physics, biology, and computer science, the book is a unique guide that offers a practical approach to the subject, including information on the basics of light-sheet fluorescence microscopy, instrumentation, applications, sample preparation, and data analysis.
The book serves as an easy-to-understand introduction to light sheet-based fluorescence, includes numerous tips and tricks for advanced users, provides detailed information on hardware and software solutions for easy implementation of light sheet fluorescence microscopy in the laboratory, and includes chapters on Alpha3 light sheet microscopes.
Written for cell biologists, biophysicists, developmental biologists, and neurobiologists, Light Sheet-based Fluorescence Microscopy provides a comprehensive overview of this microscopy technique and its latest applications.
Papers
This paper is intended to give a comprehensive review of light-sheet (LS) microscopy from an optics perspective and includes a short comparison of Alpha3 and the other light sheet microscopes. As such, emphasis is placed on the advantages that LS microscope configurations present, given the degree of freedom gained by uncoupling the excitation and detection arms. The new imaging properties are first highlighted in terms of optical parameters and how these have enabled several biomedical applications. Then, the basics are presented for understanding how a LS microscope works. This is followed by a presentation of a tutorial for LS microscope designs, each working at different resolutions and for different applications. Then, based on a numerical Fourier analysis and given the multiple possibilities for generating the LS in the microscope (using Gaussian, Bessel, and Airy beams in the linear and nonlinear regimes), a systematic comparison of their optical performance is presented. Finally, based on advances in optics and photonics, the novel optical implementations possible in a LS microscope are highlighted.
The transient receptor potential vanilloid-1 (TRPV1) ion channel is essential for sensation of thermal and chemical pain. TRPV1 activation is accompanied by Ca2+-dependent desensitization; acute desensitization reflects rapid reduction in channel activity during stimulation, whereas tachyphylaxis denotes the diminution in TRPV1 responses to repetitive stimulation. Acute desensitization has been attributed to conformational changes of the TRPV1 channel; however, the mechanisms underlying the establishment of tachyphylaxis remain to be defined. Here, we report that the degree of whole-cell TRPV1 tachyphylaxis is regulated by the strength of inducing stimulation. Using light-sheet microscopy and pH-sensitive sensor pHluorin to follow TRPV1 endocytosis and exocytosis trafficking, we provide real-time information that tachyphylaxis of different degrees concurs with TRPV1 recycling to the plasma membrane in a proportional manner. This process controls TRPV1 surface expression level thereby the whole-cell nociceptive response. We further show that activity-gated TRPV1 trafficking associates with intracellular Ca2+ signals of distinct kinetics, and recruits recycling routes mediated by synaptotagmin 1 and 7, respectively. These results suggest that activity-dependent TRPV1 recycling contributes to the establishment of tachyphylaxis.
Astrocytes are a major type of glial cell in the mammalian brain, essentially regulating neuronal development and function. Quantitative imaging represents an important approach to study astrocytic signaling in neural circuits. Focusing on astrocytic Ca2+ activity, a key pathway implicated in astrocye-neuron interaction, we here report a strategy combining fast light sheet fluorescence microscopy (LSFM) and correlative screening-based time series analysis, to map activity domains in astrocytes in living mammalian nerve tissue. Light sheet of micron-scale thickness enables wide-field optical sectioning to image astrocytes in acute mouse brain slices. Using both chemical and genetically encoded Ca2+ indicators, we demonstrate the complementary advantages of LSFM in mapping Ca2+ domains in astrocyte populations as compared to epifluorescence and two-photon microscopy. Our approach then revealed distinct kinetics of Ca2+ signals between cortical and hypothalamic astrocytes in resting conditions and following the activation of adrenergic G protein coupled receptor (GPCR). This observation highlights the activity heterogeneity across regionally distinct astrocyte populations, and indicates the potential of our method for investigating dynamic signals in astrocytes.
Titin is a giant sarcomeric protein that is involved in a large number of functions, with a primary role in skeletal and cardiac sarcomere organization and stiffness. The titin gene (TTN) is subject to various alternative splicing events, but in the region that is present at the M-line, the only exon that can be spliced out is Mex5, which encodes for the insertion sequence 7 (is7). Interestingly, in the heart, the majority of titin isoforms are Mex5+, suggesting a cardiac role for is7. Here, we performed comprehensive functional, histological, transcriptomic, microscopic and molecular analyses of a mouse model lacking the Ttn Mex5 exon (ΔMex5), and revealed that the absence of the is7 is causative for dilated cardiomyopathy. ΔMex5 mice showed altered cardiac function accompanied by increased fibrosis and ultrastructural alterations. Abnormal expression of excitation–contraction coupling proteins was also observed. The results reported here confirm the importance of the C-terminal region of titin in cardiac function and are the first to suggest a possible relationship between the is7 and excitation–contraction coupling. Finally, these findings give important insights for the identification of new targets in the treatment of titinopathies.
Three-dimensional stroma engineered models would enable fundamental and applicative studies of human tissues interaction and remodeling in both physiological and pathological conditions. In this work, we propose a 3D vascularized stroma model to be used as in vitro platform for drug testing. A pullulan/dextran-based porous scaffold containing pre-patterned microchannels of 100 μm diameter is used for co-culturing of fibroblasts within the matrix pores and endothelial cells to form the lumen. Optical clearing of the constructs by hyperhydration allows for in-depth imaging of the model up to 1 mm by lightsheet and confocal microscopy. Our 3D vascularized stroma model allows for higher viability, metabolism and cytokines expression compared to a monocultured vascular model. Stroma-endothelium cross-talk is then investigated by exposing the system to pro and antiangiogenic molecules. The results highlight the protective role played by fibroblasts on the vasculature, as demonstrated by decreased cytotoxicity, restoration of nitric oxide levels upon challenge, and sustained expression of endothelial markers CD31, vWF and VEGF. Our tissue model provides a 3D engineered platform for in vitro studies of stroma remodeling in angiogenesis-driven events, known to be a leading mechanism in diseased conditions, such as metastatic cancers, retinopathies and ischemia, and to investigate related potential therapies.
Ph.D. Theses
Matthieu Cortes
In plants, the development of aerial organs is indeterminate: it takes place throughout their lifespan. In contrast, the development of floral organs is determinate in Arabidopsis thaliana, each flower has the same number of floral organs. This difference in development is due to the maintenance or not of the pool of stem cells present in the stem cell niches, the meristems. During my thesis I showed that the transcriptional regulator VIP3 contributes to the regulation of the switch from indeterminate to determinate in flowers. This also revealed that the control of flower termination is not as robust as classically thought. Because VIP3 is also involved in the regulation of epigenetic marks and response to external mechanical stimuli, this work opens new questions on the role of mechanical signals in indeterminacy. On a more technical standpoint, the analysis of shoot development suffers from a lack of imaging methods with high temporal resolution and in-depth optical sectioning. During the last decade, light sheet microscopy has emerged as a competitive imaging modality in developmental biology. However, in plants, the technique has mainly been used in roots because of limits in the microscope design.
Svetlana Jovanic
In human, malfunctions during early developmental processes have strong implications that often compromise the survival of the embryo. We need to understand developmental processes at the level of cell behaviours and cell signalling to anticipate deleterious effects and to find potential treatments. Thanks to the high evolutionary conservation of biological processes, this can be achieved in chosen animal models and organoids. Our first goal is methodological to observe in vivo and in toto, reconstruct and quantify cell behaviours in early embryogenesis in a mammalian species – rabbit Oryctolagus cuniculus – and a non-mammalian vertebrate – zebrafish Danio rerio. Our methodology is then used to investigate fundamental cell behaviours that underlie zebrafish gastrulation in normal conditions and upon perturbations. We overcame bottlenecks to image live rabbit embryos with a prototype of an upright light sheet microscope developed by PhaseView according to our specifications. We designed 3D printed chambers for in toto imaging of mammalian early embryogenesis that allow high resolution 3D+time imaging at peri-implantation stages. Our methodology provides high quality imaging data for accurate and precise reconstruction of cell lineages and cell contours.
Tutorials
Alpha3 Dataset Visualization Workflow
PhaseView
This video shows a typical dataset visualization workflow to convert datasets acquired by Alpha3 light sheet microscope to be visualized by Imaris data visualization software.
Alpha3 Tutorials, PhaseView, 2022
Small Sample Mounting in PhaseView Alpha3 Light Sheet Microscope
PhaseView
This video demonstrates how to embed a small specimen in a well of low melting agarose and Phytagel and mount it on a stage for imaging with PhaseView Alpha3 light sheet microscope.
Alpha3 Tutorials, PhaseView, 2021
Large Sample Mounting in PhaseView Alpha3 Light Sheet Microscope
PhaseView
This video demonstrates how to mount and clamp a large specimen on a glass plate of a sample holder for imaging with PhaseView Alpha3 light sheet microscope.
Alpha3 Tutorials, PhaseView, 2021
Olympus Australia, PhaseView
This video provides a five-step overview of the start up procedure associated with the Olympus Alpha3 Light Sheet Microscope.
Alpha3 Tutorials, PhaseView Australia, 2021
Technology Talks
Data Visualization with Imaris Software
Daniel Reisen
This video shows how to visualize image datasets acquired with the Alpha3 light sheet microscope using Imaris, a versatile imaging software solution for visualization, analysis and interpretation of multidimensional microscopy data.
Alpha3 Tech Talks, Bitplane AG, 2020
Interview
Interview with Dr. Tong Wu from Evident Australia – Light microscope Alpha 3 Facility Edition. In this interview, CEO of PhaseView, Igor Lyuboshenko, presents the innovative Alpha 3 Facility Edition light sheet microscope. Interview recorded in Sydney, Olympus Australia, on May 28, 2024
PhaseView Label-free Imaging of Cell Cultures
Testing our label-free imaging unit at the Institute for Psychiatric Research in Paris, France.