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OFC - Optogenetic Flow Cytometry

Over the past years, ever since it was named 'Method of the Year 2010' by the research journal Nature Methods, optogenetics has outgrown its infancy. In the early days used mainly as a neuromodulation method, the application of optogenetic tools at present extends to many different branches of biology. For the most part and despite its many opportunities, current optogenetic in vitro research relies mostly on microscopy, patch clamp-techniques and plate readers, or using self-made and hence often inaccurate illumination devices. In addition, a major drawback of the above-mentioned methods is their inability to perform high throughput experiments at single cell resolution. A powerful tool to overcome these limitations and to revolutionize future optogenetic applications is the combination of flow cytometry with customized LED illumination – OFC.

opto biolabs white paper series - july 2020

publications using the pxONE

our pilot customers are already successfully using optogenetic flow cytometry. Some of them have already published their first stories with more to come!


optogenetic control shows that kinetic proofreading regulates the activity of the T cell receptor

The pivotal task of the immune system is to distinguish between self and foreign antigens. The kinetic proofreading model (KPR) proposes that T cells discriminate self from foreign ligands by the different ligand binding half-lives to the T cell receptor (TCR). It is challenging to test KPR as the available experimental systems fall short of only altering the binding half-lives and keeping other parameters of the ligand-TCR interaction unchanged. We engineered an optogenetic system using the plant photoreceptor phytochrome B to selectively control the dynamics of ligand binding to the TCR by light. Combining experiments with mathematical modeling we find that the ligand-TCR interaction half-life is the decisive factor for activating downstream TCR signaling, substantiating the KPR hypothesis.

Omid Sascha Yousefi, Matthias Guenther, Maximilian Hoerner, Julia Chalupsky, Maximilian Wess, Simon M. Brandl, Robert W. Smith, Christian Fleck, Tim Kunkel, Matias D. Zurbriggen, Thomas Hoefer, Wilfried Weber, Wolfgang W. A. Schamel


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LED Thermo Flow - Combining Optogenetics with Flow Cytometry

Optogenetic tools allow isolated, functional investigations of almost any signaling molecule within complex signaling pathways. A major obstacle is the controlled delivery of light to the cell sample and hence the most popular tools for optogenetic studies are microscopy-based cell analyses and in vitro experiments. The flow cytometer has major advantages over a microscope, including the ability to rapidly measure thousands of cells at single cell resolution. However, it is not yet widely used in optogenetics. Here, we present a device that combines the power of optogenetics and flow cytometry: the LED Thermo Flow. This device illuminates cells at specific wavelengths, light intensities and temperatures during flow cytometric measurements. It can be built at low cost and be used with most common flow cytometers. To demonstrate its utility, we characterized the photoswitching kinetics of Dronpa proteins in vivo and in real time. This protocol can be adapted to almost all optically controlled substances and substantially expands the set of possible experiments. More importantly, it will greatly simplify the discovery and development of new optogenetic tools.

Kathrin Brenker,  Kerstin Osthof, Jianying Yang, Michael Reth


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exciting science by our pxONE user family

we are excited about optogenetic research and proud about every scientist using our technology. Here are some research labs that just started using the pxONE.


Targeted calcium influx boosts cytotoxic T lymphocyte function in the tumour microenvironment

Adoptive cell transfer utilizing tumour-targeting cytotoxic T lymphocytes (CTLs) is one of the most effective immunotherapies against haematological malignancies, but significant clinical success has not yet been achieved in solid tumours due in part to the strong immunosuppressive tumour microenvironment. Here, we show that suppression of CTL killing by CD4+CD25+Foxp3+ regulatory T cell (Treg) is in part mediated by TGFβ-induced inhibition of inositol trisphosphate (IP3) production, leading to a decrease in T cell receptor (TCR)-dependent intracellular Ca2+ response. Highly selective optical control of Ca2+ signalling in adoptively transferred CTLs enhances T cell activation and IFN-γ production in vitro, leading to a significant reduction in tumour growth in mice. Altogether, our findings indicate that the targeted optogenetic stimulation of intracellular Ca2+ signal allows for the remote control of cytotoxic effector functions of adoptively transferred T cells with outstanding spatial resolution by boosting T cell immune responses at the tumour sites.

 

Kyun-Do Kim, Seyeon Bae, Tara Capece, Hristina Nedelkovska, Rafael G. de Rubio, Alan V. Smrcka, Chang-Duk Jun, Woojin Jung, Byeonghak Park, Tae-il Kim & Minsoo Kim


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Photopharmacology and opto-chemogenetics of TRPC channels - some therapeutic visions

Non-selective cation conductances formed by transient receptor potential canonical (TRPC) proteins govern the function and fate of a wide range of human cell types. In the past decade, evidence has accumulated for a pivotal role of these channels in human diseases, raising substantial interest in their therapeutic targeting. As yet, an appreciable number of small molecules for block and modulation of recombinant TRPC conductances have been identified. However, groundbreaking progress in TRPC pharmacology towards therapeutic applications is lagging behind due to incomplete understanding of theirmolecular pharmacology and their exact role in disease. A major breakthrough that is expected to overcomethese hurdles is the recent success in obtaining high-resolution structure information on TRPC channel complexes and the advent of TRP photopharmacology and optogenetics. Here, we summarize current concepts of enhancing the precision of therapeutic interference with TRPC signaling and TRPC-mediated pathological processes.

Sanja Curcic, Oleksandra Tiapko and Klaus Groschner


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Longitudinal proliferation mapping in vivo reveals NADPH oxidase-mediated dampening of Staphylococcus aureus growth rates within neutrophils

Upon the onset of inflammatory responses, bacterial pathogens are confronted with altered tissue microenvironments which can critically impact on their metabolic activity and growth. Changes in these parameters have however remained difficult to analyze over time, which would be critical to dissect the interplay between the host immune response and pathogen physiology. Here, we established an in vivo biosensor for measuring the growth rates of Staphylococcus aureus (S. aureus) on a single cell level over days in an ongoing cutaneous infection. Using intravital 2-photon imaging and quantitative fluorescence microscopy, we show that upon neutrophil recruitment to the infection site and bacterial uptake, non-lethal dampening of S. aureus proliferation occurred. This inhibition was supported by NADPH oxidase activity. Therefore, reactive oxygen production contributes to pathogen containment within neutrophils not only by killing S. aureus, but also by restricting the growth rate of the bacterium.

Elena A. Seiß, Anna Krone, Pauline Formaglio, Oliver Goldmann, Susanne Engelmann, Burkhart Schraven, Eva Medina & Andreas J. Müller


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Near-infrared photoactivatable control of Ca2+ signaling and optogenetic immunomodulation

The application of current channelrhodopsin-based optogenetic tools is limited by the lack of strict ion selectivity and the inability to extend the spectra sensitivity into the near-infrared (NIR) tissue transmissible range. Here we present an NIR-stimulable optogenetic platform (termed 'Opto-CRAC') that selectively and remotely controls Ca2+ oscillations and Ca2+-responsive gene expression to regulate the function of non-excitable cells, including T lymphocytes, macrophages and dendritic cells. When coupled to upconversion nanoparticles, the optogenetic operation window is shifted from the visible range to NIR wavelengths to enable wireless photoactivation of Ca2+-dependent signaling and optogenetic modulation of immunoinflammatory responses. In a mouse model of melanoma by using ovalbumin as surrogate tumor antigen, Opto-CRAC has been shown to act as a genetically-encoded 'photoactivatable adjuvant' to improve antigen-specific immune responses to specifically destruct tumor cells. Our study represents a solid step forward towards the goal of achieving remote and wireless control of Ca2+-modulated activities with tailored function.

Lian He, Yuanwei Zhang, Guolin Ma, Peng Tan, Zhanjun Li, Shengbing Zang, Xiang Wu, Ji Jing, Shaohai Fang, Lijuan Zhou, Youjun Wang,Yun Huang, Patrick G Hogan, Gang Han, Yubin Zhou


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Optogenetic Immunomodulation: Shedding Light on Antitumor Immunity.

Immunomodulatory therapies constitute a new pillar of anticancer therapy. Currently, cancer immunotherapy is associated with on-target, off-tumor cytotoxicity or immune-related adverse events. Thus, smarter immunotherapies with enhanced safety and precise control over the anticancer immune response are needed.

Optoimmunoengineering will confer light sensitivity to the immune signaling network to enable remote and noninvasive control of both innate and adaptive immune responses with high spatiotemporal precision.

Optogenetics can be made wireless by implanting miniature light-delivery devices into peripheral lymph nodes or by using red-shifted variants of optical actuators that are capable of penetrating deeper into biological tissues. Next-generation injectable, aqueous, soluble nano-optical systems are emerging for in vivo applications of optogenetics in the immune system.

Peng Tan, Lian He, Gang Han, Yubin Zhou


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more publications that we recommend

optogenetics is a fast moving research area with exciting developments on a daily basis. Here, we assemble some of our favourite papers and reviews!


Optogenetic manipulation of calcium signals in single T cells in vivo

By offering the possibility to manipulate cellular functions with spatiotemporal control, optogenetics represents an attractive tool for dissecting immune responses. However, applying these approaches to single cells in vivo remains particularly challenging for immune cells that are typically located in scattering tissues. Here, we introduce an improved calcium actuator with sensitivity allowing for two-photon photoactivation. Furthermore, we identify an actuator/reporter combination that permits the simultaneous manipulation and visualization of calcium signals in individual T cells in vivo. With this strategy, we document the consequences of defined patterns of calcium signals on T cell migration, adhesion, and chemokine release. Manipulation of individual immune cells in vivo should open new avenues for establishing the functional contribution of single immune cells engaged in complex reactions.

Armelle Bohineust, Zacarias Garcia, Béatrice Corre, Fabrice Lemaítre & Philippe Bousso 


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An Ultra-Sensitive Step-Function Opsin for Minimally Invasive Optogenetic Stimulation in Mice and Macaques

Optogenetics is among the most widely employed techniques to manipulate neuronal activity. However, a major drawback is the need for invasive implantation of optical fibers. To develop a minimally invasive optogenetic method that overcomes this challenge, we engineered a new step-function opsin with ultra-high light sensitivity (SOUL). We show that SOUL can activate neurons located in deep mouse brain regions via transcranial optical stimulation and elicit behavioral changes in SOUL knock-in mice. Moreover, SOUL can be used to modulate neuronal spiking and induce oscillations reversibly in macaque cortex via optical stimulation from outside the dura. By enabling external light delivery, our new opsin offers a minimally invasive tool for manipulating neuronal activity in rodent and primate models with fewer limitations on the depth and size of target brain regions and may further facilitate the development of minimally invasive optogenetic tools for the treatment of neurological disorders.

 

Xin Gong, Diego Mendoza-Halliday,Jonathan T. Ting, Tobias Kaiser, Xuyun Sun, André M.Bastos, Ralf D. Wimmer, Baolin Guo, Qian Chen, Yang Zhou, Maxwell Pruner, Carolyn W.-H. Wu, Demian Park, Karl Deisseroth, Boaz Barak, Edward S. Boyden, Earl K. Miller, Michael M. Halassa, Zhanyan Fu, Guoqiang Bi, Robert Desimone, Guoping Feng


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Optogenetic control of protein binding using light-switchable nanobodies

A growing number of optogenetic tools have been developed to control binding between two engineered protein domains. In contrast, relatively few tools confer light-switchable binding to a generic target protein of interest. Such a capability would offer substantial advantages, enabling photoswitchable binding to endogenous target proteins in vivo or light-based protein purification in vitro. Here, we report the development of opto-nanobodies (OptoNBs), a versatile class of chimeric photoswitchable proteins whose binding to proteins of interest can be enhanced or inhibited upon blue light illumination. We find that OptoNBs are suitable for a range of applications: modulating intracellular protein localization and signaling pathway activity and controlling target protein binding to surfaces and in protein separation columns. This work represents a first step towards programmable photoswitchable regulation of untagged, endogenous target proteins.

Agnieszka A. Gil, Evan M. Zhao, Maxwell Z. Wilson, Alexander G. Goglia, Cesar Carrasco-Lopez, José L. Avalos, Jared E. Toettcher


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Photoactivatable CRISPR-Cas9 for optogenetic genome editing

We describe an engineered photoactivatable Cas9 (paCas9) that enables optogenetic control of CRISPR-Cas9 genome editing in human cells. paCas9 consists of split Cas9 fragments and photoinducible dimerization domains named Magnets. In response to blue light irradiation, paCas9 expressed in human embryonic kidney 293T cells induces targeted genome sequence modifications through both nonhomologous end joining and homology-directed repair pathways. Genome editing activity can be switched off simply by extinguishing the light. We also demonstrate activation of paCas9 in spatial patterns determined by the sites of irradiation. Optogenetic control of targeted genome editing should facilitate improved understanding of complex gene networks and could prove useful in biomedical applications.

Yuta Nihongaki, Fuun Kawano, Takahiro Nakajima & Moritoshi Sato


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Optogenetic control of intracellular signaling pathways

Cells employ a plethora of signaling pathways to make their life-and-death decisions. Extensive genetic, biochemical, and physiological studies have led to the accumulation of knowledge about signaling components and their interactions within signaling networks. These conventional approaches, although useful, lack the ability to control the spatial and temporal aspects of signaling processes. The recently emerged optogenetic tools open exciting opportunities by enabling signaling regulation with superior temporal and spatial resolution, easy delivery, rapid reversibility, fewer off-target side effects, and the ability to dissect complex signaling networks. Here we review recent achievements in using light to control intracellular signaling pathways and discuss future prospects for the field, including integration of new genetic approaches into optogenetics.

Kai Zhang, Bianxiao Cu


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Optogenetic control of signaling in mammalian cells

Molecular signals are sensed by their respective receptors and information is transmitted and processed by a sophisticated intracellular network controlling various biological functions. Optogenetic tools allow the targeting of specific signaling nodes for a precise spatiotemporal control of downstream effects. These tools are based on photoreceptors such as phytochrome B (PhyB), cryptochrome 2, or light-oxygen-voltage-sensing domains that reversibly bind to specific interaction partners in a light-dependent manner. Fusions of a protein of interest to the photoreceptor or their interaction partners may enable the control of the protein function by light-mediated dimerization, a change of subcellular localization, or due to photocaging/-uncaging of effectors. In this review, we summarize the photoreceptors and the light-based mechanisms utilized for the modulation of signaling events in mammalian cells focusing on non-neuronal applications. We discuss in detail optogenetic tools and approaches applied to control signaling events mediated by second messengers, Rho GTPases and growth factor-triggered signaling cascades namely the RAS/RAF and phosphatidylinositol-3-kinase pathways. Applying the latest generation of optogenetic tools allows to control cell fate decisions such as proliferation and differentiation or to deliver therapeutic substances in a spatiotemporally controlled manner.

Hannes M. Beyer, Sebastian Naumann, Wilfried Weber, Gerald Radziwill

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