Optogenetics In Vitro: Illuminating Cells in Microplates

Optogenetics In Vitro: Illuminating Cells in Microplates

Amuza’s Teleopto LEDA array: an incubator compatible illumination system for optogenetics, photobiology, and photochemistry in microplates.

Several years ago we found that while more and more work in optogenetics was being done in vitro, there was little in the way of equipment specifically designed to fill this role. Many labs built their own multiwell stimulation systems. These custom designs often performed brilliantly, but they took time to design and build and often made it more difficult to share protocols with other labs. We decided to provide a light source that is reproducible from well to well and from experiment to experiment, one that is ready to use in any lab.

The LEDA arrays are the result and now cell biologists, developmental biologists, chemists, neuroscientists, and others are using this array for optogenetics and to invent new techniques.

Original LEDA-B

Our most popular LED array is the LEDA-B: it is perfectly sized for a single 96 well plate. It has 96 LEDs with emission centered at a 470 nm wavelength. A lip surrounding the array locks your plate into place so that the wells stay centered over the LEDs. It can also be used with other microplates sharing the 96 well plate footprint. The cable is 2 m long, so only the array needs to be inside the incubator.

The LAD LED driver can be controlled manually, and you can vary the irradiance from zero up to roughly 1.5 mW/mm2, depending on wavelength. You can also use low voltage TTL pulses to trigger illumination. This way you can use a programmable pulse generator to control the LED array. Lowering the voltage of the pulse lowers the voltage sent to the LED array proportionately, allowing programmatic control of not just the timing but also the intensity of illumination. Our STO pulse generator is easy to program and use, but if you already have your own pulse generator or stimulator you should be able to use it with Teleopto. We now also offer programmable multichannel  LED drivers. These drivers do not require a pulse generator, they can be programmed via software on your PC.

Our LED arrays are available in many colors to match the ever-increasing variety of light-controlled tools available in life science, including:

White 

UV 280 and 365 nm

Violet 405 and 420 nm

Blue 450 and 470 nm

Green 525 nm

Yellow / orange 590 nm

Red 630, 660, & 740 nm

Infra-Red 850 & 940 nm

If you don’t see the wavelength you need listed on the Amuza website, please let us know, we may be able to find the correct LED for your application.

Our two-color arrays have two different color LEDs under each well. For example, a blue and yellow array allows you to work with step function (bistable) options: a blue pulse of light opens the channel and a yellow pulse closes it again.

Our LEDA4 and LEDA6 arrays allow you to control four or six different sections of the plate independently. This way you can trial different intensities or pulse trains simultaneously, saving your lab time and space when developing new protocols.

We also make these arrays in different sizes so that you can illuminate multiple plates at once, or change the placement of the LEDs to match the format of different well plates. These arrays are also perfect for illuminating Petri dishes, tissue culture bottles, flasks, and other vessels.

Our LED arrays are being used for a wide range of applications, including Cry2 based biomolecular condensates, developmental biology, Stem cell differentiation, Cardiac optogenetics, receptor activation, and protein expression, oncology, ophthalmology, development of novel opsins and other optogenetic tools, photochemistry, and neuroscience.

Questions?

Wireless Optogenetics Basics

Wireless Optogenetics Basics

Introduction to Teleopto Wireless Optogenetic stimulation of mice, rats, and other small animals during behavioral testing.

Teleopto is a turnkey solution for optogenetic stimulation of mice, rats, and other small animals during behavioral experiments. Most systems use a stationary laser or LED and a long fiber optic cable for stimulation, limiting the ability of your animals to move during behavioral experiments. But since Teleopto is wireless, your research animals have complete freedom of movement and can move through doorways, tunnels, mazes, and even large open field environments.

The Teleopto system consists of remote control, wireless receiver headstages, and LED fiber-optic implants. The whole system is ready to use right out of the box. The remote sends an infrared signal to the receivers to turn the LEDs on and off. The receivers weigh as little as one gram and can be recharged between experiments. The implants are made to order with many options for light colors, single or bilateral fibers, fiber length, and fiber diameter.  Two-color implants are available for use with bistable opsins or stimulation and inhibition at the same site.

You can adjust the light power with a screwdriver. For two-channel receivers, the power for each channel is adjusted independently. Using our LPM light meter to measure the output lets you ensure that each mouse or rat in a group receives the same irradiance at the target.

Between experiments, receivers are recharged. Battery life depends on how frequently and how intensely you stimulate your mice or rats. We offer larger receivers with larger batteries for use with rats and smaller receivers for use with mice. It’s easy to switch receivers during an experiment, so chronic experiments lasting many days are possible.

Teleopto can be controlled manually, but normally a programmable pulse generator is used to send pulse trains. Our pulse generator is easy to program and use, but if you already have one it will probably be compatible with Teleopto

The pulse generator can, in turn, be triggered by behavioral events. A nose poke, lever, video tracking system, or other equipment can easily be used to trigger the start or stop of a pulse train. Amuza can work with you to make certain your behavioral equipment is compatible with Teleopto.

Teleopto is great for scaleup: a single IR emitter can control multiple receivers on multiple mice or rats. With additional emitters, one remote can be used with multiple operant conditioning chambers or cages. Additional emitters can also make certain there are no dead spots in a maze or other complex environment. The emitters have a range of 1-2 meters depending on lighting, but we also have high power IR emitters for use in large open field environments.

Questions?

Forgetfulness at the touch of a button: Teleopto Wireless Optogenetics in Science Magazine and The New York Times.

Forgetfulness at the touch of a button: Teleopto Wireless Optogenetics in Science Magazine and The New York Times.

Melanin-concentrating hormone (MCH) neurons are unlike most neurons: they are most active during sleep. Scientists have studied their role in regulating sleep and feeding behavior for some time, but the Yamanaka lab at Nagoya University in Japan has found that they may also have a role in preventing the consolidation of memories during sleep.  

Prof. Yamanaka (a co-developer of Teleopto) found that MCH neurons can suppress neurons in the hippocampus responsible for memory consolidation. His lab confirmed this role by using 

Teleopto Wireless Optogenetics: blue light and channelrhodopsin 2 were used to activate MCH neurons; green light/archaerhodopsin were used to inhibit them. This was done bilaterally during both memory consolidation (REM sleep) and awake periods. Teleopto was used so that the animals were able to move freely and interact naturally with objects during Novel Object Recognition (NOR) tests.

When mice had MCH neurons activated during sleep, their ability to remember events decreased: they forgot which objects they had encountered before sleeping and treated them the same way they treated novel objects. Conversely, when their MCH neurons were inhibited they were able to remember which objects they had already interacted with. They ignored the familiar objects and explored the novel objects instead.

When Teleopto was used to illuminate MCH neurons during awake periods, there was no effect on hippocampal-dependent memory.

 When interviewed by The New York Times, Prof. Yamanaka explained

“These results suggest that hypothalamic M.C.H. neurons help the brain actively forget new information that is not important.” And because the neurons are most active during R.E.M. sleep, they may explain why humans usually do not remember their dreams when they wake up. “The neurons may be clearing up memory resources for the next day,” Dr. Yamanaka said.

The article, “REM sleep–active MCH neurons are involved in forgetting hippocampus-dependent memories.” is available at:

https://science.sciencemag.org/content/365/6459/1308

OptoDroplets: Organelle formation controlled by Teleopto LED arrays

OptoDroplets: Organelle formation controlled by Teleopto LED arrays

What are Biomolecular condensates?

Biomolecular condensates are a unique class of organelles: they have no membranes. They can form, merge, split, and disappear in minutes, temporarily creating local incubators and assembly lines with properties very different from the bulk of the cells surrounding them. The local high concentrations of proteins and polynucleotides inside these condensates can both speed up and interfere with reactions, challenging the researchers trying to understand the rules of cell biology.  

Some condensates, such as the nucleolus and Cajal bodies, were first observed over a century ago, but others such as processing bodies, PML bodies, and paraspeckles were only discovered recently. It is only within the past few years that researchers have begun to understand that these organelles all share a common organizing principle: protein association drives the formation of gels which coalesce into the organelles themselves, which then behave according to the classic rules of phase separation and phase transition. These organelles condense in much the same way water vapor condenses into droplets on a window.

Why study biomolecular condensates?

This new understanding has led to condensates becoming a target for drug design. Dewpoint Therapeutics launched earlier this year, based on studies of stress granules. They seek to prevent temporary condensates of FUS protein from congealing into permanent aggregates, a driving force in amyotrophic lateral sclerosis (ALS). 

Liquid-liquid phase separation also has a role in gene expression: transcription factors have been found to rely on segregation inside condensates to initiate and control RNA production, yielding new targets for cancer therapies. The kinetics of ribosomal RNA processing is also proving to be dependent on the extent of gelation of the nucleolus.

Teleopto LED arrays and Biomolecular condensates

Clifford Brangwynne, Macarthur Fellow and Assoc. Prof. at Princeton University uses light to control the formation of condensates. Once activated by light, proteins like Cry2olig1 oligomerize within seconds. By fusing Cry2olig to an RNA binding protein that drives condensation in the nucleus (NPM1), the BW lab created optoDroplets: light activated condensates held together by a meshwork of protein and nucleic acids.

Blue light from a Teleopto LEDA array causes these CRY2 fusions (opto-NPM1) to coalesce into a meshwork of proteins capable of turning the nucleolus of a cell into a tightly linked gel2. The lab tunes the properties of the optoDroplets by adjusting the brightness: more light leads to more self-association and smaller pores in the meshwork. As the pores shrink, small proteins can still move through the hydrogel but larger molecules and complexes become trapped. This model allows the Brangwynne lab to study the effect of viscoelasticity on the formation of ribosomes and the processing of rRNA with just the press of a button. In a recent PNAS paper, they found that increasing the gelation of the nucleolus leads to the accumulation of larger rRNA precursors, while smaller precursors are depleted.

After the light is turned off, the condensates typically degenerate within 5 minutes. Fixing the cells while they are still illuminated allows the optoDroplets to be imaged and studied later, as shown in the figure below:

Incubator-compatible Teleopto LED arrays are tools designed for doing in-vitro optogenetics on 96 well plates. The arrays are available in wavelengths from UV to infrared and can be controlled by most pulse generators.

Postdoc Jorine Eeftens said that the Brangwynne lab used to use microscope mounted lasers to make condensates, but that only let them focus on a few cells at a time. The LEDA array allows them to activate many cells at once, greatly improving throughput in the lab. “We use it routinely, every day. We love working with it, the [LEDA] array allows us to use lots of cells, and then fix them for study. It’s our high throughput system.”

Biomolecular Condensates and Teleopto at the Woods Hole Physiology Course

The Woods Hole Marine Biology Laboratory discovery courses are intense, full-immersion summer courses for graduate students and postdocs. Students brainstorm, design and carry out their own projects – which frequently lead to publications. Ten years ago during a course led by Anthony Hyman and Brangwynne, then a postdoc in the Hyman lab, a project showed that P-granules behave like oil droplets when shearing forces are applied. The initial result from the Woods Hole class was followed up by Hyman and Brangwynne at Max Planck Institute, leading to a publication for both the students and the instructors. The paper shows that p-granule behavior follows the classic rules of phase separation and hinted at how this process could be involved in many more aspects of cellular behavior than previously thought3.

Coming full circle, this past summer Prof. Brangwynne and his postdocs led one of the Woods Hole course rotations and focused on the role of condensates in the nucleolus. They brought a LEDA array so that students could form optoDroplets in incubators during the class.

You can learn more about optoDroplets at the Brangwynne website.

Teleopto LED arrays are also being used in the development of new optogenetic switches, cardiovascular and nervous system developmental biology, ophthalmology, and photobiochemistry. 

(1) Taslimi, A., Vrana, J. D., Chen, D., Borinskaya, S., Mayer, B. J., Kennedy, M. J., & Tucker, C. L. (2014). An optimized optogenetic clustering tool for probing protein interaction and function. Nature communications, 5, 4925.

https://doi.org/10.1038/ncomms5925

(2) Zhu, L., Richardson, T. M., Wacheul, L., Wei, M. T., Feric, M., Whitney, G., … & Brangwynne, C. P. (2019). Controlling the material properties and rRNA processing function of the nucleolus using light. Proceedings of the National Academy of Sciences, 116(35), 17330-17335.

https://www.pnas.org/content/116/35/17330 

(3)Brangwynne, C. P., Eckmann, C. R., Courson, D. S., Rybarska, A., Hoege, C., Gharakhani, J., … & Hyman, A. A. (2009). Germline P granules are liquid droplets that localize by controlled dissolution/condensation. Science, 324(5935), 1729-1732.

https://doi.org/10.1126/science.1172046

 

Optogenetics Resources, Guides, and Protocols

Optogenetics Resources, Guides, and Protocols

 

We would like to share some of the many resources our customers have found useful for planning their optogenetics projects. Please post comments if you know of more resources we should include – we will update this list regularly.

Starting From Scratch

For neuroscientists, Karl Deisseroth’s Optogenetics Resource Center is perhaps the best first stop when planning your experiment. The Deisseroth lab provides optogenetics related  DNA cassettes and vectors to the optogenetic research community, as well as training and workshops for in in-vivo optogenetics on campus at Stanford University.

The Deisseroth lab (d-lab) website also hosts

  • A light power vs distance attenuation calculator for brain tissue,
  • Chromophore, DNA sequence, and vector databases,
  • Information on requesting the many viruses and DNA provided by D-lab to other researchers.
  • Links to protocols for many optogenetics based methods.

Mapping Neural Circuits

Karl Deisseroth’s 2016 Cell paper serves as both a primer on mapping neural circuits via optogenetics and a review of the many optogenetic switches available for the task.

Finding the Best Switch

If you are hunting for the right optogenetic switch for your project, the OptoBase website provides curated databases of optogenetic techniques, but the true value is in the indexing, tagging, and online tools the BIOSS team created to accelerate your search. For example, In the publication search, filters such as “multichromatic” return only papers which combine multiple optogenetic switches within a single optogenetic system and “Exclude Background” let you exclude basic research on photoreceptors.

The OptoBase’s “Find the Application” tool is a publication selector allowing you to tick off  optogenetic uses (e.g. “Control of vesicular transport” AND “control of second messengers”) and returns only those publications which actually used those methods – as opposed to just mentioning them in the discussion section. Publications are tagged and updated weekly. The Optobase is a collaborative project of the BIOSS Centre for Biological Signalling Studies.

Preventing Phototoxicity during in-vitro Experiments

Phototoxicity presents the risk of introducing artifacts or cell death in both in-vitro and in-vivo experiments, particularly when the illumination wavelength is lower than 500 nm. For in-vitro experiments,  Káradóttir et. al. found that careful choice of the culture media components can prevent many of the issues from occurring in neuronal cell cultures. In particular, removing riboflavin, thyroxine, and triiodo-1-thyronine and including additional antioxidants increases cell survival considerably.

These media and supplements are now available from Cell Guidance.

Non-neuronal Optogenetics

For non-neuronal optogenetics, EMBL has placed a short introductory course online.

Transgenic Models – Ready to Go

While many companies supply strains of mice and rats ready for transgenic manipulation to introduce optogenetic switches, the Jackson Laboratory provide many strains of transgenic mice already expressing channelrhodopsin (CHR2), archaerhodopsin (Arch), and halorhodopsin (NpHR).

Turnkey In-Vitro and wireless In-Vivo Optogenetics Systems

Amuza provides Teleopto wireless systems for in-vivo optogenetics in mice and rats as well as LED arrays for in-vitro optogenetics in culture plates.

Teleopto wireless comprises a detachable, rechargeable headstage (receiver) and LED fiber optic implants which together weigh as little as 1.3 grams. Our starter kits are turnkey solutions for your first experiment, including receivers, LED implants, remote control, charger, and stereotaxic adapters.

Teleopto LED arrays are normally made for illuminating 96 well plates inside incubators, but can be customized to many different sizes and well configurations.

Both systems are available in colors across the spectrum including UV, violet, blue, yellow, green, red, far red, and IR. Multicolor options are also available. Please visit the Amuza site for more information:

We have received an NIH Grant

We have received an NIH Grant

We are excited to announce that Amuza Inc have been awarded an R41 Technology Transfer grant by the National Institute of Neurological Disorders (NINDS/NIH, USA) to develop “a unified system of wireless optogenetics and brain microdialysis”. The grant proposal was based on research which combined Amuza’s HPLC-ECD, microdialysis, and optogenetics instruments.

The funding is for Phase I (prototype development and validation) of a Small Business Technology Transfer (STTR) project in collaboration with Drs. Nobuyoshi Suto (The Scripps Research Institute), Thomas Jhou (Medical University of South Carolina) and Matthew Buczynski (Virginia Tech).

After research and commercialization phases have  been completed the final opto-dialysis system will be available from Amuza as well. We are currently undertaking the pilot experiments in freely moving animals (both rats and mice) using our opto-dialysis prototypes. We will post the results as they become available. Please stay tuned!