$4,900.00 – $13,900.00
Optogenetic Model | S700A Fully-Auto Microtome |
|---|---|
RWD-IOS-465 | Intelligent Optogenetics System(465nm) |
RWD-IOS-589 | Intelligent Optogenetics System(589nm) |
RWD-IOS-635 | Intelligent Optogenetics System(635nm) |
Product Model | Product Description | Qty |
|---|---|---|
RWD-AFG1022 | Arbitrary Waveform/Function Generator-25MHz | 1 |
RWD-R-LG473-50-A5 | Laser (B/G/Y) | 1 |
RWD-R-FC-PC-N3-400-L1 | Patchcord | 1 |
RWD-R-FC-1x1 | Rotary Joint | 1 |
RWD-R-FC-PC-N3-400-L1 | Optical Fiber | 1 |
RWD-R-MS-1.25 | Ceramic Sleeves | 1 |
RWD-R-FOC-L200C-37NA | Ceramic Ferrule (Your Choice of size) | 1 |
RWD-R-DC-1.25 | Protective Cap for Ceramic Ferrule | 1 |
RWD-R-OFT-600 | Fiber Strippe | 1 |
RWD-R-LS-Y | Laser Goggles | 1 |
RWD-R-LP-200 | Laser Power Meter | 1 |
Optogenetics was originally used in neuroscience to describe the method of using light to image and control neuronal function. This concept has since expanded to biotechnology, merging genetic engineering and optics to control activities in intact animals. Over the last decade, optogenetics’ potential has been widely acknowledged, significantly impacting neuroscience by enabling control of specific cell types in the brain with high spatial and temporal resolution (Boyden et al., 2005). Combining optical control with detection techniques promises closed-loop control of biological systems, allowing researchers to uncover and modify intracellular signaling frameworks or multicellular dynamics to achieve desired outcomes (Grosenick et al., 2015).
Optogenetic tools require custom designs tailored to specific biological methods. Achieving optimal expression in target cell types and refining the biophysical attributes of photoreceptors are critical. Cell-type specificity is crucial, akin to genetic knock-in or knockout analyses in molecular biology. Recent advancements in single-cell analysis have shown that gene expression patterns can describe diverse cell phenotypes, making genetically encoded tools ideal for studying each cell type’s function.
Significant experiments have demonstrated optogenetics’ ability to control specific neural activities. For example, neurons activated during behavioral tasks in rodents were reactivated using light-gated ion channels, reproducing behaviors with light alone, showing optically controlled memory recall.
The use of microbial rhodopsins in mammalian neural cells exemplifies adapting natural photoreceptors for new contexts. Over 60 channelrhodopsin homologs have been discovered, enabling independent multicolor stimulation of neural populations. Additionally, light-sensitive proteins in plants and microbes have been used to control cell signaling, although optimizing these photoactivatable structures can be challenging.
Interaction upon light induction is another mode of activity in photoreceptor domains. Some domains dimerize or monomerize with light, affecting intracellular signaling and DNA transcription. Despite the modular nature of natural photoreceptors, developing new optogenetic devices requires extensive engineering. Achieving sufficient expression of optogenetic tools remains a challenge, as does optimizing multiple properties such as ion conductance, light sensitivity, and channel kinetics.
Multicolor control in optogenetics has been explored using photoreceptors with different spectral sensitivities. However, cross-activation can occur, especially with blue-light-sensitive rhodopsins. Techniques beyond spectral separation, such as leveraging differences in light sensitivity and kinetics, are needed to minimize cross-talk.
Designing studies with multiple light sources requires caution, as cross-activation can affect results. Techniques using biophysical properties of photoreceptors can reduce cross-activation, but completely eliminating small cross-talk effects may be challenging.
In summary, optogenetics holds immense potential for advancing our understanding and control of complex biological processes. Its success relies on interdisciplinary collaboration and continuous refinement of tools and techniques to address existing challenges.
Optogenetic devices often use cell-type-specific promoters, but single components limited to specific cell types are rare. Typically, cell identity involves multiple gene expressions, making this method less effective. Even available specific components might not express optogenetic tools strongly enough, and replicating endogenous gene expression involves complex regulatory elements that are unclear and hard to transport.
A common alternative is using viral vectors in transgenic animals expressing recombinase in genetically defined cells. Viral vectors like lentivirus or adeno-associated virus (AAV) support optogenetic tools under strong promoters like EF-1α. Cre-recombinase is frequently used due to the availability of many cell-type-specific Cre-transgenic lines. Recently, this method has been expanded to target neural cell types with multiple genetic markers, and viral vectors have enabled targeting based on axonal projections or synaptic connectivity (Krueger et al., 2019).
Microbial rhodopsins in mammalian neural cells use light-driven ion pumps to hyperpolarize membrane potential, producing enough current for optical silencing. The far-red-sensitive chloride pump Jaws enables noninvasive neural silencing through the mouse skull, and a light-driven outward sodium pump (KR2) is also promising. Unlike ion pumps, microbial rhodopsin ion channels control membrane potential by selective ion conduction (Boyden et al., 2005). Animal rhodopsins function as G-protein coupled receptors (GPCRs) that catalyze GDP to GTP exchange upon light activation, with some retaining their retinal chromophore, avoiding the need for supplementation.
Light-sensing proteins from plants and microbes induce light-activated conformational changes linked to other protein domains. An example is the Light-Oxygen-Voltage (LOV) domains in photoreceptor systems of plants, fungi, and bacteria. LOV domains control an effector domain fused to it by allosteric coupling or steric inhibition. For instance, the bacterial chemosensor FixL was made light-activated by replacing its PAS motif with the LOV domain Ytva (Krueger et al., 2019). This model and others show how LOV domain conformational changes can ‘uncage’ a fused effector domain.
The beginning of the dawn of optogenetics in neuroscience dates back to the ground-breaking work of Ramón y Cajal. Cajal brought forth basic proof of the fact that neurons are the signaling unit of the nervous system, and that they may be present in numerous distinguishing morphologies. From that point forward, studies were conducted to demonstrate that in fact several different kinds of neurons exist, categorized on the basis of their physiological attributes, anatomical position, morphology, and gene expression profile. The number of cells that occur in the human brain is still unclear. However, it is hypothesized that there are around 1000 neuronal cell types just inside the mammalian cortex.
Generally, it is established that a particular cell type performs the same role within a neural circuit. Consequently, the classification of all cell types and mapping out their connectivity is necessary to see how the nervous system functions. Classification of various cell types must be associated with functional identification within their regular setting, the nervous system. Therefore, neuroscientists have been searching for an approach to perturb individual cell types inside the brain.
This was clearly revealed by Francis Crick in his discussion released in 1979, in which he proposed the necessity for a technique by which all neurons of only one type could be inactivated, leaving the others essentially unaffected. Optogenetics is perhaps the first technological development that allows such experimentation. It makes the significant connection linking cell-type information and the capability to carry out gain or loss of function tests (Boyden et al., 2005). Preceding the development of the optogenetics technique, experimental viewpoints in neuroscience proposed the usage of light as a means to control neural movement.
For instance, caged compounds like the secondary messenger molecules, ions, and neurotransmitters have been created that are at first static; however, turn out to be active after being illuminated by light. Despite the fact that these photochemical ways did not present any method to control explicit cell types, they laid the foundation for the utilization of millisecond timescale illumination in cells and tissue. Precise activation of cell-type neurons was initially accomplished in a series of ground-breaking work by the Miesenböck group, by heterologously expressing invertebrate rhodopsin with additional connecting proteins and ligand-gated ion channels that can be stimulated by synthetic photocaged precursors. During this time, microbial rhodopsins that work as single component light-gated ion channels were found (Boyden et al., 2005). In mammalian neurons, these microbial photoreceptors could surprisingly be heterologously expressed to trigger action potentials with millisecond timescale accuracy optically. In fact, rhodopsins have covalently bound all-trans retinal chromophore normally created in about all cell types as well as mammalian cells and tissues (Nagel et al., 2005). The findings collectively catalyzed the wide selection of these particles in neuroscience.
The optogenetics system typically consists of the following components: waveform generator, laser (b/g/y), patchcord, rotary joint, optical fiber, ceramic sleeves, ceramic ferrule (user’s choice of size), the protective cap for the ceramic ferrule, fiber stripper, laser goggles, and laser power meter.
The waveform generator has a 20 MHz sine, and 10 MHz pulse waveforms provide coverage for most common applications. The waveform generator has a 250 MS/s sampling rate and 14-bit vertical resolution enabling the creation of high-fidelity signals (Grosenick et al., 2015). The optogenetics laser has a choice of three colors including 473nm Blu-ray Laser-50mW/power, 532nm Green Laser-50mW/power, and 593nm Yellow Laser-50mW/power. The optogenetics patchcord has low insertion loss, good repeatability, high return loss, stable temperature, and good mutual insertion performance. The optogenetics optical fiber has a choice between core diameter, ferrule size, and numerical aperture. The optogenetics rotary joint is used in awake and freely moving animals to avoid fiber optic twining.
The optogenetics ceramic cannula comes in a package of 20 and is used in a temporary connection between two optical fibers. The optogenetics ceramic ferrule has an applicable wavelength of 400-2200nm and core diameter of 100um, 200um, 300um, 400um. The optogenetics fiber stripper (tongs) is suitable for 100um-800um for stripping fiber coating and prevents damage to the fiber. The optogenetics laser goggles provide protection against the green and yellow light. The optogenetics laser power meter is applicable to multiband measurement with a broad-spectrum range and has a short response time, good thermal stability, small volume, and convenient installation.
Evaluation of Pancreatic Islets
Reinbothe & Molletuse utilized optogenetic beta-cell mouse islets for batch incubations and Ca²⁺ imaging. Mice were bred with Ins2Cre to express optogenetic proteins in beta-cells. Islets were prepared using collagenase and incubated overnight. LED illumination was applied, and blue light stimulated islets, with controls shielded from light. Post-stimulation, islets were analyzed for hormone release and Ca²⁺ levels using adjusted buffers. The setup included a fiber-coupled LED on an imaging microscope for direct stimulation, and islets were perfused with calcium imaging buffers to stabilize conditions. Optogenetic control facilitated light-induced insulin release, providing an all-optical method to regulate intracellular Ca²⁺ in beta-cells.
Evaluation of Aversive Odor Learning in Drosophila
Riemensperger, Kittel, & Fiala studied the neuronal basis of aversive olfactory learning in adult Drosophila using ChR2-XXL. They employed a barrel-type apparatus for blue light stimulation during olfactory training. Flies expressing ChR2-XXL in dopaminergic neurons were trained with synchronized odors and blue light. Post-training, flies were tested in a T-maze, and odor preferences were evaluated. Optogenetic activation mimicked the effects of a punitive shock, creating a light-induced memory associated with odors.
Evaluation of Locomotor Activity Modulation
Xu, Zhang, Guo, & Zheng used optogenetics to study locomotor activity in rats by targeting brain regions like dPAG and VTA. The procedure involved preparing optical electrodes, determining brain coordinates, and implanting the electrodes. Post-surgery, optogenetic stimulation in dPAG elicited defensive behaviors, while stimulation in VTA enhanced locomotor activity. Rats explored a behavioral field, and changes in activity were recorded, demonstrating the effects of precise brain stimulation on behavior (Brondi et al., 2022).
Evaluation of an Optogenetics Viral Vector and Optical Cannula Implantation
Pawela, DeYoe, and Pashaie used optogenetic techniques to inject an AAV virus into the rat cortex and implant an optical cannula. The process involved anesthetizing the rat, making a scalp incision, and drilling holes for viral injection. Post-injection, the skull was cleaned, and the optical cannula was implanted. Dental cement secured the cannula, and after recovery, the rat was ready for optogenetic experiments. This method allowed precise light delivery to deep brain regions, facilitating controlled neuromodulation(Pawela et al., 2016).
Evaluation of Optogenetic Approaches for Mesoscopic Brain Mapping
Kyweriga & Mohajerani combined optogenetics with voltage-sensitive dye imaging in mice to map brain functions. Transgenic mice were used with viral vectors to control optogenetic expression. The procedure involved anesthesia, craniotomy, and dye application. Brain regions were illuminated with lasers and LEDs, and imaging captured VSD activity. This method enabled detailed mapping of functional circuits and neuronal activity, providing insights into brain connectivity (Kyweriga et al., 2016).
Evaluation of Confined Stimulation in Deep Brain Structures
Castonguay, Thomas, Lesage, & Casanova developed a method using side-firing optical fibers for precise light delivery to subcortical brain areas in mice. After setting up the optical system, the mice were anesthetized, a tracheotomy was performed, and the mouse was positioned in a stereotaxic apparatus. The LGN was targeted, and neural activity was recorded. An optical fiber was inserted, and light stimulation was applied to the LGN to study neuronal responses. This technique allowed targeted optogenetic stimulation in deep brain structures for in vivo studies(Castonguay et al, 2014).
| Model | IOS-465, IOS-589, IOS-635 |
|---|
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