Single-cell resolution optogenetic stimulation of fluorescent-labeled neurons in slice using the Polygon400 (Courtsey of Dr. Blake Richards from University of Toronto).
This video shows a protein-protein interaction that is inhibited by spatially controlled blue light exposure using the Polygon400 (Courtesy of Prof. Torsten Wittman from UCSF).
The Polygon400 uses digital mirror device (DMD) technology to illuminate multiple regions simultaneously. A DMD is composed of thousands of micro-mirrors that can be individually turned on to let light pass through. Thus, you can control each mirror to control the area(s) of illumination to create any number of different sized shapes.
Simultaneous Multi-Region Illumination
High Spatial Resolution
High Frame-Rate for Pattern Switching
Low Cost & Easy to Integrate
The Polygon400 is designed to be coupled into the infinity space of any microscope model (Leica, Nikon, Olympus, Zeiss) with Mightex’s microscope-specific adapters. Please see below for different microscope coupling methods for the Polygon400, depending on the specific setup/microscope model.
The one- or three-position Polygon400 microscope adapter is inserted in between the microscope and the trinocular head of the microscope. The Polygon400 is inserted into the microscope adapter.
Compatible Models: All Models
The ring Polygon400 microscope adapter replaces the epi-illuminator on the back of the microscope. The Polygon400 is inserted into the microscope adapter.
Compatible Models: All Models
The MPI allows for both Polygon400 and a widefield illumination source (via lightguide) to be coupled to the microscope simultaneously. This solution allows users to couple a Polygon400 and widefield source to an inverted microscope, or an upright that does not have an epi-illuminator.
Compatible Models: Polygon-E, Polygon-G Series
The Polygon400 can be converted to attach to a spare camera port, if other equipment occupies the infinity space.
Compatible Models: Polygon-E, Polygon-G Series
The Polygon400 can be converted to attach to the Nikon LAPP system.
Compatible Models: Polygon-G Series
Mightex Systems offers several Polygon400 models that have been designed with different features to meet the needs of a wide-range applications that use patterned illumination. Please see below for all available Polygon400 models.
|Built-in LEDs||Lightguide-Coupled Light Source||Lightguide-Coupled Light Source||Fiber-Coupled Light Source|
|400nm – 700nm||400nm – 700nm||350nm-700nm||400nm – 700nm|
|High Power||Higher Power||Higher Power||Highest Power|
|Large Projection Area||Large Projection Area||Reduced Projection Area||Large Projection Area|
|High Resolution||High Resolution||Very High Resolution||High Resolution|
The Polygon400 DSI-E is the most cost-effective solution for patterned illumination with up to 3 LED built-in (400-700nm). This solution is useful for lower power applications, such as in vitro optogenetics.
Key Applications: Optogenetics, Photoatctivation
The Polygon400 DSI-G is a flexible solution for patterned illumination, as this patterned illuminator can be used with any lightsource (400-700nm) that accepts a 3mm core lightguide. Thus, the DSI-G provides future flexibility for different wavelengths and lightsources, depending on your application.
Key Applications: Optogenetics, Photoactivation, Photoconversion, Photopatterning
The Polygon400 DSI-DP is a flexible solution for UV patterned illumination, as this patterned illuminator can be used with any lightsource that accepts a 3mm core lightguide. Unlike the DSI-G, the DSI-DP has been optimized for UV illumination (350-700nm) applications such as, uncaging and photopatterning. In addition, the DSI-DP provides high-resolution illumination with a smaller field of view.
Key Applications: Optogenetics, Uncaging, UV Photopatterning, Photoconversion
The Polygon400 DSI-DL is a flexible solution for large field of view or high-power patterned illumination applications, as this patterned illuminator can be used with any fiber-coupled lightsource (400-700nm), such as high-power lasers. This Polygon400 model has been designed for high-power applications, such as photobleaching and in vivo optogenetics.
Key Applications: Photobleaching, Uncaging, In Vivo Optogenetics, Large-scale Optogenetics
Mightex’s PolyScan software platform is bundled with every Polygon400 to help you execute sophisticated patterned illumination experiments for your research.
The Polygon400 has been used by scientists all over the world for a wide range of research applications. Scroll through the list below to see examples of how some of our customers are using the Polygon in their research.
(a) Illustration of highly targeted optical stimulation of a single ChR2-mCherry expressing neuron in the mouse somatosensory cortex. Using a small (25 μm diameter), low-powered (3 mW) spot of illumination centred on the target cell, action potentials could be induced in current clamp (top trace), without any indication in voltage clamp of post-synaptic currents caused by spiking in other neurons (bottom trace).
(b) Illustration of illumination with a large (125 μm diameter), high-powered (15 mW) spot. Multiple spikes were induced in current clamp, and voltage clamp traces showed evidence of post-synaptic currents caused by spiking in other neurons.
Courtesy Matthew Tran & Dr Blake Richards, University of Toronto, Canada.
(A) Image of an acute brain slice prepared from a Thy1-ChR2-EYFP mouse with ChR2 expression in L5 pyramidal neurons. Whole-cell patch clamp recording from a L2/3 cell.
(B) Light-activated excitatory postsynaptic potentials (EPSPs) triggered by patterned illumination of a 10×10 grid with 473 nm LED.
(C) Colormap of the activation pattern.
(D) Histogram of automatically measured responses from all cells in a grid. Objective lens is 10X.
Courtesy Qiuyu Wu & Dr Alexander Chubykin, Purdue University, USA.
Light-evoked inhibitory postsynaptic current (IPSC) recorded in a transgenic mice expressing ChR2 in GABAergic neurons. The postsynaptic cell is non-GABAergic (ChR2 negative) and blue light stimulates GABAergic afferents expressing ChR2. Blue bars indicate the time of light illumination. Spot 1 illuminated by Polygon400 evoked reliable IPSCs whereas Spot 2 caused no response.
Courtesy Dr Wataru Inoue, University of Western Ontario, Canada.
This video shows a protein-protein interaction that is inhibited by blue light exposure. One component is coupled to the coverslip through biotin surface chemistry, the other is labeled with mCherry, and binds to the coverslip in the dark. Patterned illumination with the Polygon400 results in revesible dissociation of the mCherry-tagged protein, which rebinds within minutes of turning off blue light exposure. The Wittmann lab is working on developing this into a cell adhesion surface that can be controlled by light.
Courtesy Dr Torsten Wittmann, University California San Francisco, USA.
E18 Sprague-Dawley rat neurons were transduced with CamKII-ChR2-GFP lentivirus. Somatic activity was recorded via whole-cell patch-clamp electrophysiology. Each field was illuminated by the Polygon400 at 100% power and 20ms exposure time. Intensity of magenta pattern represents depolarization with respect to the instantaneous resting potential prior to stimulation with the Polygon’s 470nm LED. In the image, green represents the magnitude of GFP signal and black represents the fluorescence intensity of AlexaFluor 594 backfilled by the patch pipette.
Courtesy Dr Jacob Robinson, Rice University, USA.
A 50 um film of liquid tetra (ethylen glycol) diacrylate, containg 1 wt% Irgacure 819 photointiator was irradiated through a glass coverslip, using the Polygon400 and a 400nm LED light source to project a pattern onto the film surface. (A) Projection of CU logo image using the 400nm LED and a 4X objective. (B) Brightfield image of the resulting pattern in the film. Photopolymerization causes a large change in refractive index in the resin, allowing immediate visualization of the pattern. Standard development techniques could subsequently be performed by washing the film in solvent to remove the unexposed areas of liquid resin.
Courtesy Gayla Berg, University of Colorado, USA.
NMDA-receptor mediated synaptic currents recorded at a +40mV membrane potential were elicited by light activation of channelrhodopsin-expressing terminals of thalamocortical afferents onto an upper layer cortical GABAergic interneuron. moving the 470nm rectangular illumination to the right or the left of the cell activates inputs of different strength innervating different somatodendritic domains of the cell. In black and red are the individual and the average traces respectively.
Courtesy Dr. Theofanis Karayannis, University of Zurich, Switzerland.
A) Light-evoked response of a head-fixed larva expressing channelrhodopsin (right). Photostimulation site was indicated by a blue circle (470nm).
B) A schematic diagram of the Polygon400 patterned illumination in in vivo optogenetic mapping system.
Courtesy Dr. Sachiko Tsuda, Saitama University, Japan.
An example of connectivity mapping that allow to reproduce some results from in Valera et al. (elife, 2016). 100um RuBi glutamate was uncaged at various locations in the granular layer with 20ms pulses (blue bar), exciting notably granule cells. We can then measure the spatial organization of the granule cells to Purkinje cell (PC) connections by recording PCs in whole cell patch clamp. Granule cells triggers both monosynaptic excitatory current onto PCS (left map, measured a -60mW), but also disynaptic inhibitory currents via molecular layer interneurons (right map, measured at 0 mW). Average evoked responses at one location (dotted blue square) are shown at the bottom of the figure. Data storage, measurements, and map representations can be made online, using a homemade software in python.
Courtesy, Dr. Antoine Valera & Dr. Angus Silver, University College London, UK.
A) Patch-clamp recording of the current generated by the channelrhodopsin when activated with the blue LED light from the Polygon400 illuminator as indicated by the blue bar.
B) Activation of an action potential (upper trace) and the corresponding current under voltage-clamp conditions (lower trace). The action potential could be evoked by a 0.5ms stimulation of the blue LED light from the Polygon400 illuminator at 60% intensity.
Courtesy, Dr. Hans van Hooft, University of Amsterdam, Netherland.
The first figure a repetitive stimulation with a larger circular pattern which elicited increasing responses until an action potential is generated. The second figure is increasing the intensity of stimulation from 30% to 100% and keeping the size of the pattern the same. Here we’re driving the cell at 10Hz at 100% and one can see that the stimulation is sufficient to drive doublets of action potentials during each bout of depolorization.
Courtesy, Dr. Geoffrey G. Murphy, University of Michigan, USA.
A) Acute brain slice from a YFP-channelrhodopsin-2 (ChR-2) mouse depicting its expression in cortical L5 pyramidal neurons. B) L5 pyramidal neuron from the somatosensory cortex filled with Alexa 594 to allow visualization of neuronal compartments without stimulating ChR-2. Blue dots indicate on the illuminated areas (Blue LED – 470 nm) along the apical dendrite. Expansion of the marked areas depicting the delicate dendrites that were stimulated. C) Electrophysiological (patch clamp) current recordings from the soma, corresponding to local photostimulation of ChR-2 by blue light. The numbers in the bottom of the trace corresponds to the stimulated dot numbers as indicated in B.
Courtesy, Dr. Yossi Buskila, University of Western Sydney, Australia.
Purkinje cell (PC) firing is monitored, while various light stimulation patterns are delivered to the granular layer (gl) and/or molecular layer (ml) of acute mouse cerebellar slices. The mice cerebellum was injected with AAVs carrying the ChR2 construct. The figure shows the firing frequency increase caused in a PC unit (scale bar 500ms) by optogenetic activation of a granular layer ROI (blue rectangle).
Courtesy, Dr. Lisa Mapelli & Dr. Simona Tritto, University of Pavia, Italy.
Feola, I., Volkers,L., Majumder, R., Teplenin, A., Schalij, M.J., Panfilov, A.V., Vries, A.A.F., & Pijnappels, D.A. “Localized Optogenetic Targeting of Rotors in Atrial Cardiomyocyte Monolayers”. Arrhythmia and Electrophysiology, 10(11) (2017).
Wilson, M.Z., Ravindran, P.T., Lim, W.A., & Toettcher, J.E. “Tracing Information Flow from Erk to Target Gene Induction Reveals Mechanisms of Dynamic and Combinatorial Control”. Molecular Cell, 67(5), 757-769 (2017).
Guo , Z.X., Ling, J.C., Ai, L.L., Zhou, W., Gong, X., Yong, M.Z., Xiong, L.Y., & Shi, J.W. “Transgene is specifically and functionally expressed in retinal inhibitory interneurons in the VGAT-ChR2-EYFP mouse”. Neuroscience, 363, 107-119 (2017).
Andrasi, T., Verses, J.M., Rovira-Esteban, L., Kozma, R., Vikor, A., Gregori, E., & Hajos, N. “Differential excitatory control of 2 parallel basket cell networks in amygdala microcircuits”. PLoS Biology (2017).
Shimizu, K. & Stopfer, M. “A Population of Projection Neurons that Inhibits the Lateral Horn but Excites the Antennal Lobe through Chemical Synapses in Drosophila”. Frontiers in Neural Circuits (2017).
Malyshev, A.Y., Roshchin, M.V., Smirnova, G.R., Dolgikh, D.A., Balaban, P.M., & Ostrovsky, M.A. “Chloride conducting light activated channel GtACR2 can produce both cessation of firing and generation of action potentials in cortical neurons in response to light”. Neuroscience Letters, 640, 76-80 (2017).
Duan, J.G., Fu, H., & Zhang, J.Y. “Activation of Parvalbumin-Positive Neurons in Both Retina and Primary Visual Cortex Improves the Feature-Selectivity of Primary Visual Cortex Neurons”. Neuroscience Bulletin, 33(3), 255–263 (2017).
Watanabe, M., Feola, I., Majumder, R., Jangsangthong, W., Teplenin, A.S., Ypey, D.L., Schalij, M.J., Zeppenfeld, K., Vries, A.A.F., & Pijnappels, D.A. “Optogenetic manipulation of anatomical re-entry by light-guided generation of a reversible local conduction block”. Cardiovascular Research, 113(3), 354–366 (2017).
Fiedler, C.I., Aisenbrey, E.A., Heveran, C.M., Ferguson, V.L., Bryant, S.J., & McLeod, R.R. “Enhanced mechanical properties of photo-clickable thiol-ene PEG hydrogels through repeated photopolymerization of in-swollen macromer”. Soft Matter, 44 (2016).
Butler, J.L., Mendonça, P.R. F., Robinson, H.P.C., & Paulsen, O. “Intrinsic Cornu Ammonis Area 1 Theta-Nested Gamma Oscillations Induced by Optogenetic Theta Frequency Stimulation”. Journal of Neuroscience, 36(15), 4155-4169 (2016).
García, N.V.D.M., Priya, R., Tuncdemir, S.N., Fishell, G., & Karayannis, T. “Sensory inputs control the integration of neurogliaform interneurons into cortical circuits”. Nature Neuroscience 18, 393–401 (2015).
Peng, H.Y., Wang, C., Xi, W.X., Kowalski, B.A., Gong, T., Xie, X.L., Wang, W.T., Nair, D.P., McLeod, R.R., & Bowman, C.N. “Facile Image Patterning via Sequential Thiol–Michael/Thiol–Yne Click Reactions”. Chemistry of Materials, 6819–6826 (2014).
This white paper provides an overview of the molecular probes available to the cell biologist and how they have been applied in different fields, going over specific examples from modulation of intracellular signaling pathways to organelle positioning dynamics. It also compares common patterned illumination technologies that cell biologists are familiar with: galvo-based scanning and digital micromirror device (DMD) systems. Read more.
Are you interested in utilizing patterned illumination in your neuroscience research and deciding what tool is right for you?
This paper will discuss several techniques of patterned illumination and their relative merits in an experimental setting, specifically the use of direct projection systems such as digital micromirror devices (DMD), holographic projection systems, and galvo-based scanning systems in combination with optogenetic constructs. Each of these techniques has its own advantages and drawbacks. Read more.
Are you interested in all-optical methods for probing in vivo neural activity and deciding what tool is right for you?
This paper will discuss several tools that enable in vivo calcium imaging and optogenetics and their relative merits in an experimental setting. Specifically, the advantages and drawbacks of two-photon imaging, head-mounted microscopes, fiber photometry, and fiberscopes for in vivo imaging and optogenetics will be discussed. Read more.