Name: building a simple and versatile illumination system for optogenetic experiments
Uploaded: 2021-01-12T15:00:00.000+00:00
Duration: 6 min 41 s
Description: Watch this Scientific Journal Video about Building a Simple and Versatile Illumination System for Optogenetic Experiments at JoVE.com

We would like to thank Yingxiao (Peter) Wang, Ziliang Huang, and Molly Allen for testing out different versions of the LED system as it was being developed. This work was supported by the Kavli Institute for Brain and Mind at UC San Diego and the Salk Institute, National Science Foundation through the NSF Center for Science of Information under Grant CCF-0939370, NIH Grant NS060847, and NIH Grant R21DC018237.

1. Build the electrical circuit
NOTE: The protocol for building a single circuit for an available LED is described here. Instructions to expand this up to four LEDs are included in the supplementary info.
<ol>
	<li>Turn on the smoke absorber and the soldering iron. Add water to the wiping sponge, have the solder at hand. 
	CAUTION: Make sure to take safety precautions to remove smoke and prevent burns.</li>
	<li>Begin soldering circuit components to the printed circuit board (PCB board) in the order shown in the supplementary panels. 
	NOTE: Use a small amount of solder on the soldering iron tip to heat the metal of the component and the PCB board first and melt additional solder directly onto the components; flux can help a lot.</li>
	<li>Solder jumper wires and components (Supplementary Figure 2 and Supplementary Figure 3).
	<ol>
		<li>For the jumper wires (the insulated wiring that connects two points on the circuit board), use two pieces of orange [7.6 mm (0.3 inch)] and yellow [12 mm (0.4 inch)] wires from the jumper kit.</li>
		<li>Clip the PCB board onto the &#8220;helping hands&#8221; and insert the jumper wires in the following pinholes, bend the terminals 45 degrees and add flux (Figure 2, Supplementary Figure 2 and Supplementary Figure 3): a1 and a3 &#8594; ground (-) (orange), a7 &#8594; power supply (+) #7 (yellow), d2 &#8594; d6 (yellow).</li>
		<li>Solder and then trim the back of the wires.</li>
		<li>Insert LM317T voltage regulator in the following pinholes, bend the pins, and add flux (Figure 2 and Supplementary Figure 4): Adj &#8594; e5, Vout &#8594; e6, Vin &#8594; e7.</li>
		<li>Solder the left and right terminals first, trim them, then solder and trim the middle terminal.</li>
		<li>To set the low voltage range of the circuit, insert an 820 &#8486; resistor all the way down into pinholes, solder and trim c2 &#8594; c5 (Figure 2 and Supplementary Figure 5).</li>
		<li>To enable LED control by the microcontroller, insert the transistor into b3&#8211;b5 (Figure 2 and Supplementary Figure 6): Collector &#8594; b3, Base &#8594; b4, Emitter &#8594; b5. 
		NOTE: Be aware of the orientation of the transistor to insert correctly; check the specs to find the Collector, Base, and Emitter designation.</li>
	</ol>
	</li>
	<li>Solder the wire-to-wire connectors for the POT, LED, microcontroller, and power source. 
	NOTE: Pay attention to the color of the wires of the wire-to-wire connectors and whether using a female or male wire-to-wire connector.
	<ol>
		<li>Determine whether a &#8220;low voltage&#8221; circuit or &#8220;high voltage&#8221; circuit is required for the desired LED (Figure 1C). 
		NOTE: If the LED is on the &#8220;low voltage&#8221; list, a resistor in parallel with the POT is required.</li>
		<li>For the &#8220;low voltage&#8221; or &#8220;high voltage&#8221; circuit, place the wire from a female wire-to-wire connector through hole a5 (Supplementary Figure 7). Do not solder into place yet if making the low voltage circuit. 
		NOTE: Twist the bare wire ends so that the small wire hairs don&#8217;t flake out. If the wire seems too thick to push through the pinhole without fraying, cut 2&#8211;6 strands and then twist them back together (Supplementary Figure 7B&#8211;D).</li>
		<li>If making the &#8220;high voltage&#8221; circuit, skip to step 1.4.5. If making the &#8220;low voltage&#8221; circuit, push a 560&#8486; resistor through the same hole (a5) and solder with the wire-to-wire connector lead.</li>
		<li>Connect the other end of the resistor to the ground (Supplementary Figure 7G).</li>
		<li>Insert the other end of the female wire-to-wire connector soldered into a5 hole connecting it to the ground and solder it (Supplementary Figure 8A,B).</li>
		<li>For the microcontroller connection, insert one end of a male wire-to-wire connector into hole a4 and the other into a hole connected to the ground (Supplementary Figure 9A&#8211;C).</li>
		<li>For the LED connection, insert one end of a female wire-to-wire connector into hole a2 and the other end into a hole connected to the ground (Supplementary Figure 9D,E).</li>
	</ol>
	</li>
</ol>
2. Build power supply, manual power switch, POTs, and microcontroller connection
<ol>
	<li>Build the power supply.
	<ol>
		<li>Solder an orange [7.6 mm (0.3 inch)] jumper from a29 to the ground (Supplementary Figure 10).</li>
		<li>Solder a female wire-to-wire connector from a30 to the power supply (+) (Supplementary Figure 11A&#8211;C).</li>
		<li>Solder a male wire-to-wire connector from c29 to c30 (Supplementary Figure 11D&#8211;F).</li>
		<li>Cut the connector off a power supply cord, expose the wires, and strip them (Supplementary Figure 12A&#8211;C).</li>
		<li>Add flux to the wires prior to soldering using a flux pen (Supplementary Figure 3G).</li>
		<li>Place a 3.18 mm (1/8 inch) shrink tube around a male wire-to-wire connector and a thicker piece 4.76 mm (3/16 inch) over the power supply wire (Supplementary Figure 12D).</li>
		<li>Twist the wires from the power supply and the male wire-to-wire connector together and solder (Supplementary Figure 12E, 13A,B).</li>
		<li>Place the smaller diameter shrink tube 3.18 mm (1/8 inch) over the connections and shrink them with a heat gun (Supplementary Figure 13C,D).</li>
		<li>Place a larger diameter shrink tube 4.76 mm (3/16 inch) over the smaller shrink tube 3.18 mm (1/8 inch) and heat again (Supplementary Figure 13E,F).</li>
	</ol>
	</li>
	<li>Build the manual power switch.
	<ol>
		<li>Place the shrink tube 3.18 mm (1/8 inch) over the wires for the switch (Supplementary Figure 14A).</li>
		<li>Twist and solder the wires of a male wire-to-wire connector (Supplementary Figure 14B,C).</li>
		<li>Place the shrink tube 3.18 mm (1/8 inch) over soldered sections and shrink with a heat gun (Supplementary Figure 14D,E).</li>
	</ol>
	</li>
	<li>Connect the male wire-to-wire connector to the POT.
	<ol>
		<li>Loop the wire-to-wire connector&#39;s black wire around the middle terminal of the POT (Supplementary Figure 15B).</li>
		<li>Twist the wire that is looped tightly around the terminal and solder it (Supplementary Figure 15C). 
		NOTE: Small precision pliers can assist in making a tight twist.</li>
		<li>Repeat with the red wire connection to the terminal, as in Supplementary Figure 15D.</li>
		<li>Use pliers to break the metal tab near the red arrow (Supplementary Figure 15E,F).</li>
	</ol>
	</li>
	<li>Build the microcontroller connection (only necessary for computer-controlled LEDs).
	<ol>
		<li>If making a LED driver for more than one LED, cut off the black wires from all but one female wire-to-wire connector (Supplementary Figure 16A).</li>
		<li>Crimp the ends of the wire-to-wire connectors, as shown (Supplementary Figure 16B&#8211;D).</li>
		<li>Push the crimped ends through the rectangular connector (Supplementary Figure 16E).</li>
	</ol>
	</li>
</ol>
3. Build the LEDs
<ol>
	<li>Strip the wire ends (~5 mm) and apply flux using a flux pen as in Supplementary Figure 3G. 
	NOTE: To efficiently solder the wires onto the LED base, flux must be added to the contacts on the LED base and the wires.</li>
	<li>Tin the wire by heating the wire from below and adding solder from the top (Supplementary Figure 17B).</li>
	<li>Use the flux pen to place flux onto the surface contact of the LED base (Supplementary Figure 17C).</li>
	<li>Place a generous amount of solder onto a large soldering tip (~4&#8211;5 mm) (Supplementary Figure 17D), use it to heat the LED base at the contact (Supplementary Figure 17E). After a few seconds, drag the solder across the contact (Supplementary Figure 17F). Repeat steps 3.3&#8211;3.4 on the other contact (Supplementary Figure 17G). 
	CAUTION: The LED base can get very hot during soldering. Place the LED base on a surface that will not melt or burn.</li>
	<li>Clip the black wire onto the contact &#8220;C+&#8221; (the cathode) using the hair clips (Supplementary Figure 18A).</li>
	<li>Place a generous amount of solder onto the large soldering tip (Supplementary Figure 18B) and press it down on the wire until the solder on the LED base melts (Supplementary Figure 18C). Hold down the wire (Supplementary Figure 18D) and remove the soldering iron while holding the wire in place (Supplementary Figure 18E).</li>
	<li>Place a small amount of solder paste onto the pads for the LED connections (Supplementary Figure 19A,B) and place the LED over the pads using forceps (Supplementary Figure 19C). 
	NOTE: If the placement is a little off, it is okay; it will go into place once the solder paste melts.</li>
	<li>Hold the red wire on the &#8220;A+&#8221; (anode) and clip it with a hair clip (Supplementary Figure 20A&#8211;C).</li>
	<li>Place a generous amount of solder onto the large soldering tip (Supplementary Figure 20D) and press it down onto the wire until the solder on the LED base and the solder paste under the LED melts (Supplementary Figure 20E). 
	NOTE: After the solder paste melts, the color becomes silver (Supplementary Figure 20H,I).</li>
	<li>Choose the length of the wire needed for the desired set up. Strip the LED wires, and a male wire-to-wire connector (Supplementary Figure 21A) then add flux as in Supplementary Figure 3G.</li>
	<li>Place the shrink tube over the wires. Use a 3.18 mm (1/8 inch) shrink tube over the wire-to-wire connectors and a 4.76 mm (3/16 inch) shrink tube over the wire (Supplementary Figure 21B).</li>
	<li>Clip the wire-to-wire connector with a &#8220;helping hands&#8221; and twist the connector end with the wire (Supplementary Figure 21C) and solder them. Repeat with the other wire (Supplementary Figure 21D,E).</li>
	<li>Place the 3.18 mm (1/8 inch) shrink tubes over the solder and shrink (Supplementary Figure 21F&#8211;G).</li>
	<li>Place the 4.76 mm (3/16 inch) shrink tube over the 3.18 mm (1/8 inch) shrink tube and shrink (Supplementary Figure 21H&#8211;I).</li>
	<li>Clip the LED wires the &#8220;helping hands&#8221; with tape under it (Supplementary Figure 22A).</li>
	<li>Mix epoxy according to the manufacturer&#8217;s instructions and spread over the top of the soldered LED (Supplementary Figure 22B). Leave overnight to cure.</li>
	<li>If mounting using a touch fastener, cut a small piece of the touch fastener (Supplementary Figure 23A) and press it against the back of the LED for 30 s.</li>
	<li>Use a high-speed rotary tool to make a notch on the lid of a black box (Supplementary Figure 23C&#8211;E).</li>
	<li>Build a mounting for a single LED through a privacy film.
	<ol>
		<li>Using the spade drill bit, drill a 1.75 cm (11/16 inch) hole through the top of a black box where the LED will be placed (Supplementary Figure 24A).</li>
		<li>Using a high-speed rotary tool, make a notch on one side of the hole to make room for the LED wire, as shown in Supplementary Figure 24A.</li>
		<li>Cut a piece of privacy film (25&#8211;30 mm) and tape onto the inside of the black box covering the hole that the LED will illuminate through (Supplementary Figure 24A).</li>
		<li>Place the LED outside the black box on top of the hole with privacy film and tape in place with electrical tape (Supplementary Figure 24B&#8211;E).</li>
	</ol>
	</li>
</ol>
4. Accommodate a black box to fit the illumination system
<ol>
	<li>For a four LED system, drill four 0.83 cm (21/64 inch) holes on the lid 3.81 cm (1.5 inch) apart where the potentiometers will be attached (Supplementary Figure 25).</li>
	<li>Using a high-speed rotary tool, cut a 1.19 cm x 1.90 cm (0.47 inch x 0.75 inch) rectangular hole in the top-left corner (Supplementary Figure 25).</li>
	<li>Using the spade drill bit, drill a 1.75 cm (11/16 inch) hole on the black box (Supplementary Figure 26).</li>
	<li>File the holes and insert the grommet into the hole drilled in (Supplementary Figure 26).</li>
	<li>For the computer-controlled LEDs, sandpaper the area where the microcontroller will be glued in a black box, as well as the bottom side of the microcontroller holder.</li>
	<li>Snap the microcontroller onto the holder before securing the holder in the black box and then epoxy them in place (Supplementary Figure 27A).</li>
	<li>Use sandpaper to sand the bottom of two clips and the area in a black box where the circuit will be placed and secure the clips inside the black box with the epoxy (Supplementary Figure 27A).</li>
	<li>Secure the PCB board into clips (Supplementary Figure 27B).</li>
	<li>Push the power switch through the square hole in the lid made in Supplementary Figure 25 and snap it into place (Supplementary Figure 28A).</li>
	<li>Push the POTs through the holes on the lid, screw into place (Supplementary Figure 28A), and put the knob onto the POT (Supplementary Figure 28B).</li>
</ol>
5. Connect all of the wiring and devices
<ol>
	<li>Label the wire-to-wire connectors (e.g., LED, POT, COM) (Supplementary Figure 29A).</li>
	<li>Attach the crimped connectors made in step 2.4 (Supplementary Figure 16) to the male wire-to-wire connector between the two female connectors (POT and LED) (Supplementary Figures 7A and S37).</li>
	<li>Connect the crimped ends into the microcontroller (Supplementary Figure 30).</li>
	<li>Pull the USB cable through the grommet and plug it into the microcontroller.</li>
	<li>Pull the wires for the LEDs through the grommet and connect to the female wire-to-wire connector on the left of the microcontroller connection (Supplementary Figures 9D and 38).</li>
	<li>Pull the wire for the power supply through the grommet and connect it to the male wire-to-wire connector on the right side of the PCB board (Supplementary Figure 11D).</li>
	<li>Connect the male wire-to-wire connector from the power switch to the female wire-to-wire connector on the right of the PCB board (Supplementary Figure 11A).</li>
	<li>Connect the male wire-to-wire connectors from the POTs on the lid to the female wire-to-wire connectors on the PCB board (Supplementary Figures 8 and 36). 
	NOTE: Do not turn on the circuit without the potentiometers connected.</li>
</ol>
6. Install the LED control software
NOTE: See the detailed Software Installation Instructions in the supplementary file on Github. https://github.com/BreakLiquid/LED-Control-User-Interfaces
<ol>
	<li>Download and install the software for programming the microcontroller</li>
	<li>Download and install the package manager.</li>
	<li>Program the microcontroller.</li>
	<li>Download and install the runtime engine.</li>
	<li>Download the user interface.</li>
</ol>
7. Stimulate the cells with light
<ol>
	<li>Transfect HEK293 cells.
	<ol>
		<li>Plate HEK293 cells at 100k cells per well in a 24-well plate.</li>
		<li>Use the example table to calculate serum-free media, Polyethylenimine (PEI), and DNA volumes (Supplementary Figure 39) and transfect using the manufacturer&#8217;s protocol.</li>
	</ol>
	</li>
	<li>Stimulating cells with light. 
	NOTE: Cells must be kept in the dark after transfection or handled using a light source that does not excite the optogenetic system.
	<ol>
		<li>Decide what type of stimulation will be used on the cells (continuous light, pulsing intensity, etc.).</li>
		<li>With the POTs turned off (counterclockwise), turn on the LED power supply.</li>
		<li>Place a light meter inside the black box where the cells will be placed and place the lid with the LED over the meter. Adjust the light intensity as needed.</li>
		<li>If using the computer to control the LEDs, open the user interface software.</li>
		<li>Program the user interface (Figure 5A,B).
		<ol>
			<li>On the top left panel, select the COM port for the microcontroller and click on Connect.</li>
			<li>Use the panels on the right to program each LED. For continuous light, select any time except for zero in the &#8220;Time On&#8221; and set the &#8220;Time Off&#8221; to zero.</li>
			<li>On the bottom right panel, program the main timing control.
			<ol>
				<li>To delay illumination, select a start delay (HH:MM).</li>
				<li>To shut off all LEDs after a designated time, select a run time (HH:MM).</li>
				<li>Start the illumination program by clicking on the Run button (Figure 5A).</li>
			</ol>
			</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
8. Measure gene expression using a dual luciferase assay
<ol>
	<li>Prepare luciferase reagent by mixing 10 mL of luciferase buffer with luciferase reagent and aliquot in 1 mL tubes to be stored at -80 &#176;C for up to 1 year.</li>
	<li>Prepare lysis buffer 5x into 1x for 100 &#181;L for N + 2 wells. e.g., for 30 samples, 30 x 20 &#181;L of 5X lysis buffer, and 30 x 80 &#181;L of MQ H2O.</li>
	<li>Prepare Renilla substrate solution: 20 &#181;L of Renilla substrate for 1 mL of Renilla buffer (this amount is suitable for 10 assays).</li>
	<li>Remove cells from the incubator, aspirate the media, add 100 &#181;L of 1x lysis buffer per well and place it on a shaker at 100 RPM for 15 min.</li>
	<li>Place in -20 &#176;C for at least 1 h.</li>
	<li>Add 100 &#181;L of luciferase reagent per sample into a well of a white 96-well plate.</li>
	<li>Set up the plate reader for luminescence. Using the luminometer module of the plate reader, set the integration for 1 s.</li>
	<li>Add thawed lysates in wells below the luciferase reagent. Using a multichannel pipette, mix 20 &#181;L of sample into the luciferase reagent and measure luminescence immediately.</li>
	<li>After the readings plateau, add 100 &#181;L of Renilla substrate solution and scan again.</li>
	<li>Divide the Luciferase signal by the Renilla signal to account for transfection efficiency.</li>
	<li>Compare luciferase signals normalized for transfection efficiency (e.g., compare the signal from red light illuminated and far-red light illuminated samples).</li>
</ol>

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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+blue-light-mediated+induction+of+protein+interactions+in+living+cells.">Rapid blue-light-mediated induction of protein interactions in living cells.</a> Nature Methods. 7 (12), 973-975 (2010).</li><li>Taslimi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+second-generation+CRY2-CIB+dimerizers+and+photoactivatable+Cre+recombinase.">Optimized second-generation CRY2-CIB dimerizers and photoactivatable Cre recombinase.</a> Nature Chemical Biology. 12 (6), 425-430 (2016).</li><li>Shimizu-Sato, S., Huq, E., Tepperman, J. M., Quail, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+light-switchable+gene+promoter+system.">A light-switchable gene promoter system.</a> Nature Biotechnology. 20 (10), 1041-1044 (2002).</li><li>M&#252;ller, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+red/far-red+light-responsive+bi-stable+toggle+switch+to+control+gene+expression+in+mammalian+cells.">A red/far-red light-responsive bi-stable toggle switch to control gene expression in mammalian cells.</a> Nucleic Acids Research. 41 (7), 77 (2013).</li><li>Levskaya, A., Weiner, O. D., Lim, W. A., Voigt, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatiotemporal+control+of+cell+signalling+using+a+light-switchable+protein+interaction.">Spatiotemporal control of cell signalling using a light-switchable protein interaction.</a> Nature. 461 (7266), 997-1001 (2009).</li><li>Levskaya, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+biology:+engineering+Escherichia+coli+to+see+light.">Synthetic biology: engineering Escherichia coli to see light.</a> Nature. 438 (7067), 441-442 (2005).</li><li>Kaberniuk, A. A., Shemetov, A. A., Verkhusha, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+bacterial+phytochrome-based+optogenetic+system+controllable+with+near-infrared+light.">A bacterial phytochrome-based optogenetic system controllable with near-infrared light.</a> Nature Methods. 13 (7), 591-597 (2016).</li><li>Redchuk, T. A., Omelina, E. S., Chernov, K. G., Verkhusha, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Near-infrared+optogenetic+pair+for+protein+regulation+and+spectral+multiplexing.">Near-infrared optogenetic pair for protein regulation and spectral multiplexing.</a> Nature Chemical Biology. 13 (6), 633-639 (2017).</li><li>Ong, N. T., Olson, E. J., Tabor, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+an+E.+coli+near-infrared+light+sensor.">Engineering an E. coli near-infrared light sensor.</a> ACS Synthetic Biology. 7 (1), 240-248 (2018).</li><li>Zhang, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetic+control+with+a+photocleavable+protein,+PhoCl.">Optogenetic control with a photocleavable protein, PhoCl.</a> Nature Methods. 14 (4), 391-394 (2017).</li><li>Lee, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporally+precise+labeling+and+control+of+neuromodulatory+circuits+in+the+mammalian+brain.">Temporally precise labeling and control of neuromodulatory circuits in the mammalian brain.</a> Nature Methods. 14 (5), 495-503 (2017).</li><li>Kim, M. 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The authors have no conflicts of interest to disclose.

The LED system described here has been used in our lab to optimize, characterize, and work with several optogenetic tools. In Kyriakakis et al.4, we tested many combinations of PhyB-PIF gene switches in parallel. We then used this system to test pulses of light at different frequencies to measure the gene switch kinetics and effective light intensity. This system was also used to optimize and characterize two optogenetic systems that use blue light for stimulation5,6. Since only one LED needed to be bright enough to activate most optogenetic tools, buying a system with large numbers of LEDs over each well is not always necessary. This setup is inexpensive, reliable, easy to reconfigure, and requires no prior electrical expertise to follow the assembly protocol.
In the supplementary Supplementary Figures 31&#8211;38, we describe how to incorporate up to four LEDs into the system. While this may limit some experiments requiring a large number of parallel conditions, more LEDs can be added by replacing the 9 Volt power supply used in this protocol with a higher wattage one. Similarly, several lower power LEDs can be connected in parallel to each circuit. In this latter arrangement, some LEDs will not be controlled individually, but this can be useful when many LEDs are required to cover a larger area. Once familiar with the electronics of this system, there are many ways to customize it. Additional strategies for customizing the system include placing the LED farther or closer to the sample and illuminating through filters/diffusers for homogenous illumination conditions or to prevent heating as in (Supplementary Figure 23) and Allen et al.5. Another notable feature of our LED design is that it is encapsulated in epoxy and has a touch fastener on the back; this allows the LED to be securely placed with ease virtually anywhere: in incubators, fish tanks, animal cages, walls, etc.
Many experiments that use optogenetics to control genes, signaling pathways, and other cellular activity often require pulsing, span large time scales, or need to be performed in an incubator, therefore requiring automatization or remote manipulation without a microscope. This LED system has been tested continuously for several months inside a humidified CO2 incubator without any issues. Additionally, with reversible systems such as PhyB optogenetic systems, the experimenter may need to program-specific pulsing illumination schedules. In our previous work4, we used pulsing programs to test the reversibility dynamics of a PhyB-PIF3 switch in mammalian cells through the user interface. Using the methodology described in this manuscript, programming a pulsing protocol is easy, providing the flexibility and autonomy needed for many types of optogenetic experiments in a user-friendly manner.
The most critical steps in building this system include putting together the electrical circuit on the PCB board and connecting the components, which are detailed in section 1 and section 2. It is essential to carefully follow each step in these sections and double-check the pinhole numbers line-by-line before soldering each component. Section 2 explains how to set up the components that will be connected to the circuit. So that the components connect in the correct orientation, it is particularly important to ensure that the colors of the black and red wires on the wire-to-wire connectors match. Small oversights in these two sections will very likely affect the system&#8217;s functionality. Indeed, the first step in troubleshooting this method will be to check that the circuit was built correctly and that all the connections are in place. Secondly, checking the soldering quality for loose connections and the wires for flaring wirehairs that may be shorting the circuit is of particular importance. A third step would be to ensure that the LEDs are working correctly, which can be done using a power supply or a 1.5 V battery by clipping the LED&#39;s two terminals with alligator clips. Another potentially critical consideration is to prevent heating (when using the LEDs at high power) or diffusing the light for wider spread illumination. To address these considerations, the LEDs can be mounted outside of a black box with &#8220;privacy film&#8221; on the inside, as described in Supplementary Figure 23 and Allen et al.5. Because of the simplicity of this system, taking it apart to verify, modify, upgrade, or repair modular components is not difficult.
Another critical factor for inducible gene systems is to consider how much activation is required or how much leakiness is acceptable for the biological system being controlled. As shown in Figure 6, these can vary with the amount of reporter DNA. In addition, the transfection efficiency and, therefore, the copy number of reporter constructs in each cell will vary. It may be advantageous for some experiments to make a cell line with a fixed amount of reporter or PhyB gene-switch components and screen for clones with the desired range of induced expression, as is commonly done with drug inducible systems. Due to the size and instability of lentiviral plasmid pPK-2304, we also made non-lentiviral plasmid versions of the PhyB switch in the pcDNA backbone pPK-351 (Addgene #157921) and pPK-352 (Addgene #157922).
By building this LED illumination system following this protocol, users have all the components necessary to perform a wide array of optogenetics experiments in vitro and in vivo. Combined with the instructions for using PhyB-PIF3 in mammalian cells, this protocol will allow non-engineers and biologists to, flexibly and effectively, use PhyB-based optogenetics in a variety of contexts.

Optogenetic tools are becoming ubiquitous and new technology is constantly being developed to optically control biological processes such as gene expression, cell signaling, and many more1,2,3. The ability to control cellular processes with light allows for fast kinetics, tight spatial control, and dose-dependent regulation that can be controlled by light intensity and exposure time. To use these tools, a device to control these parameters is necessary. We have recently developed a genetically encoded PhyB-PIF3 mammalian gene switch that reversibly activates and deactivates genes using red/far-red light, respectively4. This system was tested in several mammalian cell lines and enabled the unparalleled induction of gene expression even with very small amounts of light, including pulses of light. Researchers wishing to use the PhyB switch and similar tools5,6 frequently request information on methods to control illumination intensity and duration. Therefore, we developed this protocol with step-by-step instructions to enable broader adoption of these tools for optogenetics.
Before the widespread use of LEDs, broadband light sources with filters were used to study light-responsive proteins such as phytochromes7. Recently, some LED illumination systems have been published along with optogenetic tools8,9,10,11,12, but those protocols can require significant expertise in electronics/software, require specialized equipment (e.g., 3D printers, laser cutting machines, or photomasks), or do not provide the step-by-step instructions some researchers would need to deploy for their research needs. While independent control of individual wells in a multiwell plate can be useful, it is often unnecessary when researchers only need to compare several different samples in light and dark or red light versus far-red light. Also, many existing commercial systems are expensive, with limited customization capability. However, the LEDs described in this protocol are cost-effective, bright, and can be mounted in many ways; therefore, they can be used to illuminate several different types of samples. With the protocol and the software provided, LEDs ranging from ultraviolet (UV) to NIR can be used and controlled with software to perform optogenetic experiments using UVR813,14, Dronpa15,16, LOV domains17,18, Step Function Opsins19,20, CRY221,22, PhyB4,23,24,25, bacterial phytochromes26,27,28,29 and other light-responsive systems30,31,32.
This protocol constitutes a tutorial for the assembly of the circuits and other hardware needed to control different parameters for light stimulation as well as the molecular/cellular tools to run an optogenetic experiment. In addition, we report plasmids optimized from Kyriakakis et al.4 that are smaller and more stable for cloning. Through this protocol, biologists without expertise in electronics and optics can build illumination systems that are flexible and robust. In a step-by-step fashion, we show how to build LED systems, removing the technical bottleneck for the broader adoption of optogenetic tools. This system can easily be used in most cell culture incubators, even if they do not contain wire ports. For example, we have kept the LED system in a humidified CO2 incubator continuously for more than 6 months with no decrease in performance. We also explain how to connect the LED system to a computer and interface it with open-source software we provide on GitHub (https://github.com/BreakLiquid/LED-Control-User-Interfaces). Building a system using this protocol provides researchers the basic knowledge to debug potential issues, replace parts, and improve/extend functionalities.
System overview
Building the illumination system involves (1) building the electronic circuit, (2) building the peripherals (power supply cord, power switch, etc.), (3) building the LEDs, (4) assembling all of these components, and (5) installing the software to control the LEDs with a user interface (Figure 1A). Once completed, the illumination system can control up to four LEDs independently with a user interface (Figure 1B). The user interface enables each LED to pulse at specified time intervals and shut off after a specified time. There is also a start-delay to begin illumination programs at a specified time. Potentiometers (POTs) regulate each LED&#8217;s intensity independently or can be used for manual LED control without a computer. The wires to the LEDs can be any custom length, allowing them to be easily placed in an incubator or lab space. Due to these LEDs&#8217; high power, they can be used to illuminate a large area with a single LED from a distance.
LED driver description
To power and control the LEDs&#39; intensity, this protocol will go through steps to build an &#8220;LED driver&#8221;. Each LED has a range of voltages in which it operates (Figure 1C). During operation, the regulator&#39;s output voltage, which controls the light intensity, can be tuned by a potentiometer. The POT varies the resistance, adjusting the output voltage/brightness. Tuning with a 1k&#937; (1 kilo-ohm) POT gives what we call the &#8220;high-voltage circuit&#8221; and has a range of 1.35 V to 2.9 V. Since 2.9 V is too high for operating the lower voltage LEDs (Figure 1C), we show a single modification (Resistor 3 or &#8220;R3&#8221; Supplementary Figure 1A) that limits the range to match the low voltage LEDs. R3 serves to decrease the maximum voltage applied to the LEDs to 1.85 V (assembly detailed in Supplementary Figure 8) when in parallel with the potentiometer. By using voltage to control brightness instead of current, the system is more flexible for LEDs with different operating voltages. Figure 1C contains a list of the high and low voltage LEDs to guide the optimal circuit selection. This design keeps the minimum voltage low enough so that the LED is completely off when the potentiometer is off and does not allow the voltage to go above the typical operating voltage of the LED. For PhyB optogenetics, we use deep red and far-red LEDs, which use the low-voltage circuit.
LED computer control system description
The LED illumination system can be used for constant illumination without a computer or microcontroller. However, for pulsing programs and for controlling individual LED timing, a microcontroller must be installed. To use a microcontroller to control the LEDs, a transistor is required to connect the microcontroller to the circuit. This transistor senses voltage from the microcontroller and switches from being conductive or insulating. To control the &#8220;on&#8221; and &#8220;off&#8221;, we use what is called an &#8220;NPN switching type transistor&#8221; (2N2222) as a controllable shunt across R2 (Supplementary Figure 1A). When the voltage from the microcontroller is applied to the transistor base, the transistor becomes conductive and makes the LED voltage low, turning the LED off. Thus, the LED and transistor on and off states are directly controlled by the microcontroller, which is controlled by the software installed on the PC.
For making the illumination system, the following steps are required: Build the electrical circuit; build the power supply, manual power switch, POTs, and microcontroller connection; build the LEDs; accommodate a black box to fit the illumination system; connect all of the wiring and devices; install the LED control software, stimulate the cells with light; measure gene expression using a dual luciferase assay.

<table><tbody><tr><td>18AWG 2pin RED Black wire</td><td>Amazon</td><td>15M-28AWG-2468</td><td>Inexpensive wire to connect LEDs to the power circuit.&lt;br/&gt; https://www.amazon.com/gp/product/B072KGYH1M/ref=oh_aui_detailpage_o05_s00?ie=UTF8&amp;amp;psc=1</td></tr><tr><td>1K Ohm potentiometer</td><td>Amazon</td><td>52161500</td><td>2 x 1K Ohm potentiometer potential + 2 x black control Knob.&lt;br/&gt; https://www.amazon.com/gp/product/B00XIWA2GO/ref=oh_aui_detailpage_o00_s00?ie=UTF8&amp;amp;psc=1</td></tr><tr><td>20 Gauge Silicone JST Connector</td><td>Amazon</td><td>SIM&amp;amp;NAT 5.9 inch 2 Pin Male Female JST RCY Plug Connectors</td><td>These are very common and there are many equivalents.&lt;br/&gt; https://www.amazon.com/gp/product/B071XN7C43/ref=oh_aui_detailpage_o00_s01?ie=UTF8&amp;amp;psc=1</td></tr><tr><td>22 AWG solid jumper wires</td><td>Amazon</td><td>WJW-60B-R</td><td>Jameco Valuepro WJW-60B-R Wire Jumper Kit 350 each 22 AWG, 14 Lengths 10 Colors 25 Of Each Length.&lt;br/&gt; https://www.amazon.com/Jameco-Valuepro-WJW-60B-R-Jumper-Lengths/dp/B01KHWEB3W/ref=sr_1_5?s=industrial&amp;amp;ie=UTF8&amp;amp;qid=1519261370&amp;amp;sr=1-5&amp;amp;keywords=solid+wire+breadboard&amp;amp;dpID=51UopZhPJeL&amp;amp;preST=_SX342_QL70_&amp;amp;dpSrc=srch</td></tr><tr><td>560 ohm 1/2watt 1% tolerance</td><td>Amazon</td><td>a14051600ux0301</td><td>Uxcell a14051600ux0301 60 Piece Axial Lead 1% Tolerance Colored Ring Metal Film Resistor Resistance, 560 Ohm 1/2W.&lt;br/&gt; https://www.amazon.com/a14051600ux0301-Tolerance-Colored-Resistor-Resistance/dp/B016ZU2DGC/ref=pd_day0_328_9?_encoding=UTF8&amp;amp;pd_rd_i=B016ZU2DGC&amp;amp;pd_rd_r=XTM6KHQ3NT8DHWB1QWZN&amp;amp;pd_rd_w=txGNx&amp;amp;pd_rd_wg=ELyii&amp;amp;psc=1&amp;amp;refRID=XTM6KHQ3NT8DHWB1QWZN</td></tr><tr><td>820 ohm 1/2watt 1% tolerance</td><td>Amazon</td><td>TTL-A-8035-50Ea</td><td>Set of 50Ea Metal Film Resistor 820 Ohm 1% 1/2W (0.5W).&lt;br/&gt; https://www.amazon.com/50Ea-Metal-Film-Resistor-0-5W/dp/B00VGU2SS0/ref=sr_1_14?s=industrial&amp;amp;ie=UTF8&amp;amp;qid=1518045187&amp;amp;sr=1-14&amp;amp;keywords=1%2F2W+820+Ohm+resistor</td></tr><tr><td>A Male to B Male Cable (10 Feet)</td><td>Amazon</td><td>Part# 30-001-10B</td><td>The cable that comes with the Arduino doesn&#039;t fit well in the box.&lt;br/&gt; https://www.amazon.com/gp/product/B001MSU1HG/ref=oh_aui_detailpage_o07_s00?ie=UTF8&amp;amp;psc=1</td></tr><tr><td>Ardiuino UNO equivilent</td><td>Amazon</td><td>Elegoo EL-CB-001</td><td>UNO R3 Board ATmega328P ATMEGA16U2 with USB Cable for Arduino.&lt;br/&gt; https://www.amazon.com/gp/product/B01EWOE0UU/ref=oh_aui_detailpage_o03_s00?ie=UTF8&amp;amp;psc=1</td></tr><tr><td>Arduino holder</td><td>Digikey</td><td>X000018</td><td>Fits very snug.&lt;br/&gt; https://www.digikey.com/product-detail/en/arduino/X000018/1050-1150-ND/8135632</td></tr><tr><td>Black boxes for circuits and light chambers</td><td>Amazon</td><td>1591ESBK</td><td>Hammond 1591ESBK ABS Project Box Black.&lt;br/&gt; https://www.amazon.com/gp/product/B0002BSRIO/ref=oh_aui_detailpage_o07_s00?ie=UTF8&amp;amp;psc=1</td></tr><tr><td>Blue LED</td><td>Digikey</td><td>LXML-PB01-0040</td><td>LED LUXEON REBEL BLUE SMD. Uses &quot;Saber 20 mm Star base&quot;&lt;br/&gt; https://www.digikey.com/product-detail/en/lumileds/LXML-PB01-0040/1416-1029-1-ND/3961134</td></tr><tr><td>Cable ties</td><td>Amazon</td><td>sd027</td><td>Tarvol Nylon Zip Ties (Pack of 100) 8 Inch with Self Locking Cable Ties (White).&lt;br/&gt; https://www.amazon.com/Tarvol-Nylon-Locking-Cable-White/dp/B01MRD0JRR/ref=sr_1_7?s=hi&amp;amp;ie=UTF8&amp;amp;qid=1519261882&amp;amp;sr=1-7&amp;amp;keywords=Cable+ties&amp;amp;dpID=51zUNmuUjyL&amp;amp;preST=_SY300_QL70_&amp;amp;dpSrc=srch</td></tr><tr><td>Command Fridge Clips</td><td>Amazon</td><td>17210CLR</td><td>Clips for holding circuit board inside of the black box. Command strips can also be used.&lt;br/&gt; https://www.amazon.com/gp/product/B0084M69YM/ref=oh_aui_detailpage_o00_s01?ie=UTF8&amp;amp;psc=1</td></tr><tr><td>Cyan LED</td><td>Digikey</td><td>LXML-PE01-0070</td><td>LED LUXEON REBEL CYAN SMD. Uses &quot;Saber 20 mm Star base&quot;&lt;br/&gt; https://www.digikey.com/products/en?keywords=1416-1031-1-nd</td></tr><tr><td>Electrical tape - 3M Scotch #35 Electrical Tape Value Pack</td><td>Amazon</td><td>03429NA</td><td>Scotch 700 Electrical Tape, 03429NA, 3/4 in x 66 ft.&lt;br/&gt; https://www.amazon.com/Scotch-Electrical-Tape-4-Inch-66-Foot/dp/B001ULCB1O/ref=psdc_256161011_t1_B001B19FDK</td></tr><tr><td>Farred LED 720nm</td><td>Luxeon Star LEDs</td><td>LXML-PF01</td><td>Far Red (720nm) LUXEON Rebel LED. Uses &quot;Saber 20 mm Star base&quot;&lt;br/&gt; https://www.luxeonstar.com/lxml-pf01-far-red-luxeon-rebel-led-260mW</td></tr><tr><td>Farred LED 740nm</td><td>Ushio</td><td>EDC740D-1100-S5</td><td>Uses &quot;STAR XP 3535&quot; base&lt;br/&gt; https://www.ushio-optosemi.com/jp/products/led/power/pdfs/edc/EDC740D-1100-S5.pdf</td></tr><tr><td>Farred LED 780nm</td><td>Ushio</td><td>EDC780D-1100</td><td>Uses &quot;STAR XP 3535&quot; base&lt;br/&gt; http://www.ushio-optosemi.com/jp/products/led/power/pdfs/edc/EDC780D-1100.pdf</td></tr><tr><td>Farred LED 810nm</td><td>Ushio</td><td>EDC810D-1100</td><td>Uses &quot;STAR XP 3535&quot; base&lt;br/&gt; http://www.ushio-optosemi.com/jp/products/led/power/pdfs/edc/EDC810D-1100.pdf</td></tr><tr><td>Farred LED 850nm</td><td>Ushio</td><td>EDC850D-1100</td><td>Uses &quot;STAR XP 3535&quot; base&lt;br/&gt; http://www.ushio-optosemi.com/jp/products/led/power/pdfs/edc/EDC850D-1100.pdf</td></tr><tr><td>Grommets</td><td>Amazon</td><td>Pico 6120D</td><td>These are very common and there are many equivalents.&lt;br/&gt; https://www.amazon.com/Pico-6120D-Vinyl-Grommets-Package/dp/B0002ZG47G</td></tr><tr><td>Hair/Alligator Clips</td><td>Amazon</td><td></td><td>1-3/4 Inch (45 Mm)- Hair Clips Single Prong Metal Alligator Clips Hairbow Accessory -Silver,50 Pcs.&lt;br/&gt; https://www.amazon.com/gp/product/B00K09T3L8/ref=oh_aui_detailpage_o00_s00?ie=UTF8&amp;amp;psc=1</td></tr><tr><td>LED base</td><td>Luxeon Star LEDs</td><td>LXB-RS20A</td><td>Saber 20 mm Star Blank Aluminum MCPCB Base For Rebel LEDs&lt;br/&gt; https://www.luxeonstar.com/saber-20mm-star-blank-mcpcb-base-for-a-rebel-leds</td></tr><tr><td>LED PCB fopr Ushio LEDs</td><td>Adura LED solutions</td><td>STAR XP 3535 Package LED</td><td>Fits many other LEDs by Ushio&lt;br/&gt; http://aduraled.com/product/pcb/1901-star-xp-3535-package-led</td></tr><tr><td>Loctite Epoxy Clear Multi-Purpose, 0.85-Fluid Ounce Syringe</td><td>Amazon</td><td>1943587</td><td>Loctite Epoxy Clear Multi-Purpose, 0.85-Fluid Ounce Syringe.&lt;br/&gt; https://www.amazon.com/Loctite-Multi-Purpose-0-85-Fluid-Syringe-1943587/dp/B011INNBN0/ref=psdc_256243011_t4_B0044FBB8C</td></tr><tr><td>NTE Heat Shrink 2:1 Assorted Colors and Sizes 160 PCS</td><td>Amazon</td><td>B000FIDTYG</td><td>These are very common and there are many equivalents.&lt;br/&gt; https://www.amazon.com/NTE-Heat-Shrink-Assorted-Colors/dp/B000FIDTYG/ref=sr_1_1?s=hi&amp;amp;ie=UTF8&amp;amp;qid=1519261282&amp;amp;sr=1-1&amp;amp;keywords=nte+shrink&amp;amp;dpID=41L5l7LCfiL&amp;amp;preST=_SX342_QL70_&amp;amp;dpSrc=srch</td></tr><tr><td>Picture Hanging Velcro Strips</td><td>Amazon</td><td>PH204-16NA</td><td>With these you can hang the LEDs in many places.&lt;br/&gt; https://www.amazon.com/Command-Picture-Hanging-16-Pairs-PH204-16ES/dp/B073XS3CHV/ref=pd_sim_60_5?_encoding=UTF8&amp;amp;pd_rd_i=B073XS3CHV&amp;amp;pd_rd_r=112KGQJNRRYD0RAT598H&amp;amp;pd_rd_w=3n34Q&amp;amp;pd_rd_wg=sRvec&amp;amp;psc=1&amp;amp;refRID=112KGQJNRRYD0RAT598H</td></tr><tr><td>Power supply</td><td>Amazon</td><td>tb013</td><td>Any other 9V 1.5Z AC/DC converter will do becuase we cut the end off anyway.&lt;br/&gt; https://www.amazon.com/gp/product/B06Y1LF8T5/ref=oh_aui_detailpage_o07_s01?ie=UTF8&amp;amp;psc=1</td></tr><tr><td>Power switch Rocker Switch</td><td>Amazon</td><td>SIXQJZML</td><td>These are very common and there are many equivalents.&lt;br/&gt; https://www.amazon.ca/COOLOOdirect-Solder-Rocker-Switch-Toggle/dp/B071Y7SMVQ/ref=sr_1_31?_encoding=UTF8&amp;amp;c=ts&amp;amp;dchild=1&amp;amp;keywords=Boat+Rocker+Switches&amp;amp;qid=1594434474&amp;amp;s=sports&amp;amp;sr=1-31&amp;amp;ts_id=2438617011</td></tr><tr><td>Rectangular Connectors - for crimped wires</td><td>Digikey</td><td>2183-1905-ND</td><td>6 Rectangular Connectors - Housings Black 0.100&quot; (2.54mm)&lt;br/&gt; https://www.digikey.com/product-detail/en/pololu-corporation/1905/2183-1905-ND/10450382?utm_adgroup=Rectangular%20Connectors%20-%20Housings&amp;amp;utm_source=google&amp;amp;utm_medium=cpc&amp;amp;utm_campaign=Shopping_Product_Connectors%2C%20Interconnects_NEW&amp;amp;utm_term=&amp;amp;utm_content=Rectangular%20Connectors%20-%20Housings&amp;amp;gclid=Cj0KCQjwvIT5BRCqARIsAAwwD-QmETT-ko07ote5VQgodKvWU0uDG8GYN7Vj-6WVBBOWdSgPaPd9azAaAhVLEALw_wcB</td></tr><tr><td>Red LED</td><td>Digikey</td><td>LXM3-PD01</td><td>LED LUXEON REBEL DEEP RED SMD. Uses &quot;Saber 20 mm Star base&quot;&lt;br/&gt; https://www.digikey.com/products/en?keywords=1416-1701-1-nd</td></tr><tr><td>Sandpaper</td><td>Amazon</td><td>B002NEV6GS</td><td>3M Wetordry Sandpaper, 03022, 800 Grit, 3 2/3 inch x 9 inch.&lt;br/&gt; https://www.amazon.com/3M-03022-Imperial-Wetordry-Sandpaper/dp/B002NEV6GS/ref=sr_1_1?dchild=1&amp;amp;keywords=3M+Imperial+Wetordry+3-2%2F3+in.+x+9+in.+800+Grit+Sandpaper+Sheets+%2810+Sheets-Pack%29&amp;amp;qid=1594435012&amp;amp;sr=8-1</td></tr><tr><td>Solder for soldering wires and circuit components</td><td>Amazon</td><td>Mudder Lead Free Solder Wire Sn99 Ag0.3 Cu0.7 with Rosin Core for Electrical Soldering 0.22lbs (0.6 mm)</td><td>These are very common and there are many equivalents.&lt;br/&gt; https://www.amazon.com/Mudder-Solder-Electrical-Soldering-0-22lbs/dp/B01B61TWGY</td></tr><tr><td>Solder-able Breadboard for building the circuit</td><td>Amazon</td><td>GK1007</td><td>Gikfun Solder-able Breadboard Gold Plated Finish Proto Board PCB Diy Kit for Arduino (Pack of 5PCS) GK1007.&lt;br/&gt; https://www.amazon.com/gp/product/B071R3BFNL/ref=oh_aui_detailpage_o04_s00?ie=UTF8&amp;amp;psc=1</td></tr><tr><td>Spade drill bit</td><td>Amazon</td><td>Irwin 88811</td><td>These are very common and there are many equivalents.&lt;br/&gt; https://www.amazon.com/Speedbor%C2%AE-Blue-GrooveTM-Standard-Length-Woodboring/dp/B017S9JXB2/ref=sr_1_10?ie=UTF8&amp;amp;qid=1519516560&amp;amp;sr=8-10&amp;amp;keywords=11%2F16+spade+bit</td></tr><tr><td>Transistor</td><td>Newark</td><td>2N2222A</td><td>Can buy from many places.&lt;br/&gt; http://www.newark.com/nte-electronics/2n2222a/bipolar-transistor-npn-40v-to/dp/10M4197</td></tr><tr><td>Voltage regulator</td><td>Newark</td><td>LM317T</td><td>Equivilent to NTE956.&lt;br/&gt; https://www.newark.com/stmicroelectronics/lm317t/adjustable-linear-regulator-1/dp/89K0685?gclid=CjwKCAiAu9vwBRAEEiwAzvjq-1rmUi6lvOIFFt-BxttHXvmAeUoni4NM0BW-BtM_LMliSqxA9Xq4KxoCfikQAvD_BwE&amp;amp;mckv=sQqHZDDRz_dc|pcrid|219869297712|plid||kword|lm317t|match|p|slid||product||pgrid|35966450488|ptaid|kwd-541160713|&amp;amp;s_kwcid=AL!8472!3!219869297712!p!!g!!lm317t&amp;amp;CMP=KNC-GUSA-SKU-MDC</td></tr><tr><td>Windows 10 tablet</td><td>Amazon</td><td>B08BYTT79Y</td><td>Any Windows 10 PC will do.&lt;br/&gt; https://www.amazon.com/gp/product/B08BYTT79Y/ref=ppx_yo_dt_b_asin_title_o01_s00?ie=UTF8&amp;amp;psc=1</td></tr><tr><td>&lt;strong&gt;Cell Culture Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Human Embryonic Kidney 293 cells HEK293</td><td>ATCC</td><td>ATCC CRL-1573</td><td>Common Cell line.&lt;br/&gt; https://www.atcc.org/products/all/CRL-1573.aspx</td></tr><tr><td>Fetal Bovine Serum</td><td>ThermoFisher</td><td>26140079</td><td>These are very common and there are many equivalents.&lt;br/&gt; https://www.thermofisher.com/order/catalog/product/26140079#/26140079</td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle Medium High Glucose</td><td>ThermoFisher</td><td>11965&amp;minus;092</td><td>These are very common and there are many equivalents.&lt;br/&gt; https://www.thermofisher.com/order/catalog/product/11965118?SID=srch-srp-11965118#/11965118?SID=srch-srp-11965118</td></tr><tr><td>10,000 units/mL of penicillin and 10,000 &amp;micro;g/mL of streptomycin</td><td>ThermoFisher</td><td>15140122</td><td>These are very common and there are many equivalents.&lt;br/&gt; https://www.thermofisher.com/order/catalog/product/15140122?SID=srch-srp-15140122#/15140122?SID=srch-srp-15140122</td></tr><tr><td>White Corning 96-Well Solid Black or White Polystyrene Microplates</td><td>ThermoFisher</td><td>07-200-589</td><td>White plates are preferred. Do not use clear plates.&lt;br/&gt; https://www.fishersci.com/shop/products/costar-96-well-black-white-solid-plates-8/p-152852</td></tr><tr><td>PEI MAX - Transfection Grade Linear Polyethylenimine Hydrochloride (MW 40,000)</td><td>PolySciences</td><td>24765-1</td><td>Can be replaced with another transfection reagent.&lt;br/&gt; https://www.polysciences.com/default/catalog-products/life-sciences/transfection-reagents/polyethylenimine-max-mw40000-high-potency-linear-pei/</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name of Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Diagonal Cutting Plier (110mm)</td><td>Amazon</td><td>Proskit 1PK-037S</td><td>These are very common and there are many equivalents.&lt;br/&gt; https://www.amazon.com/iExcell-Diagonal-Cutting-Nippers-Chrome-Vanadium/dp/B076XYVS6Y/ref=sr_1_11?dchild=1&amp;amp;keywords=diagonal+cutting+pliers&amp;amp;qid=1594436230&amp;amp;sr=8-11</td></tr><tr><td>Dremil 3000 with cutting tool and grinder</td><td>Amazon</td><td>Dremel 3000</td><td>Dremel 3000-2/28 Variable Speed Rotary Tool Kit- 1 Attachments &amp;amp; 28 Accessories- Grinder, Sander, Polisher, Router, and Engraver.&lt;br/&gt; https://www.amazon.com/Dremel-3000-2-28-Attachments-Accessories/dp/B005JRJE7Y/ref=sr_1_3?dchild=1&amp;amp;keywords=Dremel+200-1%2F15+Two-Speed+Rotary+Tool+Kit&amp;amp;qid=1594436404&amp;amp;s=hi&amp;amp;sr=1-3</td></tr><tr><td>Dremil cutting and grinding tool</td><td>Amazon</td><td>Dremel 200-1/15</td><td>Any similar Dremil will work.&lt;br/&gt; https://www.amazon.com/Dremel-200-1-Two-Speed-Rotary-Tool/dp/B002BAHF8W/ref=sr_1_1?s=hi&amp;amp;ie=UTF8&amp;amp;qid=1519268058&amp;amp;sr=1-1&amp;amp;keywords=dremel+200&amp;amp;dpID=41h9ZucnTYL&amp;amp;preST=_SY300_QL70_&amp;amp;dpSrc=srch</td></tr><tr><td>Dremil grinding tip</td><td>Amazon</td><td>Dremel 84922</td><td>Silicon Carbide Grinding Stone.&lt;br/&gt; https://www.amazon.com/Dremel-84922-Silicon-Carbide-Grinding/dp/B00004UDKD/ref=sr_1_fkmr0_1?s=hi&amp;amp;ie=UTF8&amp;amp;qid=1519268585&amp;amp;sr=1-1-fkmr0&amp;amp;keywords=dremel+tip+84922</td></tr><tr><td>EDSYN The Original Deluxe SOLDAPULLT</td><td>Amazon</td><td>DS017</td><td>For removing solder/mistakes.&lt;br/&gt; https://www.amazon.com/EDSYN-The-Original-Deluxe-SOLDAPULLT/dp/B006GOKVKI</td></tr><tr><td>Helping Hand with Magnifying Glass</td><td>Amazon</td><td>SE MZ101B</td><td>These are very common and there are many equivalents.&lt;br/&gt; https://www.amazon.com/SE-MZ101B-Helping-Magnifying-Glass/dp/B000RB38X8/ref=sr_1_4?s=hi&amp;amp;ie=UTF8&amp;amp;qid=1519268108&amp;amp;sr=1-4&amp;amp;keywords=Helping+hands&amp;amp;dpID=31GEhMw7WvL&amp;amp;preST=_SX300_QL70_&amp;amp;dpSrc=srch</td></tr><tr><td>Pointed Nose Micro Pliers</td><td>Amazon</td><td>Hakko CHP PN-20-M</td><td>Steel Super Specialty Pointed Nose Micro Pliers with Smooth Jaws, 1.0mm Nose.&lt;br/&gt; https://www.amazon.com/Hakko-PN-20-M-Specialty-Pointed-Pliers/dp/B00FZPGUBI/ref=sr_1_1?s=hi&amp;amp;ie=UTF8&amp;amp;qid=1519268153&amp;amp;sr=1-1&amp;amp;keywords=Hakko+CHP+PN-20-M+Steel+Super+Specialty+Pointed+Nose+Micro+Pliers+with+Smooth+Jaws%2C+1.0mm+Nose&amp;amp;dpID=3109XRgwn3L&amp;amp;preST=_SX342_QL70_&amp;amp;dpSrc=srch</td></tr><tr><td>Small screw drivers</td><td>Amazon</td><td>Wiha 26197</td><td>These are very common and there are many equivalents.&lt;br/&gt; https://www.amazon.com/26197-Precision-Slotted-Phillips-Screwdrivers/dp/B01L46TEN2/ref=sr_1_1?s=hi&amp;amp;ie=UTF8&amp;amp;qid=1519268018&amp;amp;sr=1-1&amp;amp;keywords=Wiha+precision+set</td></tr><tr><td>Soldering iron</td><td>Amazon</td><td>Yihua 939D+ Digital Soldering Station</td><td>These are very common and there are many equivalents.&lt;br/&gt; https://www.amazon.com/Professional-Digital-Soldering-Station-Switch/dp/B07YSCBZ4F/ref=psdc_13837391_t1_B07RVMZNYR</td></tr><tr><td>TraceTech No-Clean Flux Pen</td><td>Amazon</td><td>2507-N</td><td>Tech Spray 2507-N No-Clean Flux Dispensing Pen, 11.5 mL.&lt;br/&gt; https://www.amazon.com/Tech-Spray-2507-N-No-Clean-Dispensing/dp/B00DDF2FYS/ref=sr_1_1?dchild=1&amp;amp;keywords=2507-N&amp;amp;qid=1595469618&amp;amp;sr=8-1</td></tr><tr><td>Weller WSA350 120v Bench Top Smoke Absorber</td><td>Amazon</td><td>WSA350</td><td>For soldering safety.&lt;br/&gt; https://www.amazon.com/Weller-WSA350-Bench-Smoke-Absorber/dp/B000EM74SK</td></tr><tr><td>Wire strippers</td><td>Amazon</td><td>CSP-30-7</td><td>These are very common and there are many equivalents.&lt;br/&gt; https://www.amazon.com/Hakko-CSP-30-7-Stripper-Maximum-Capacity/dp/B00FZPHY7M/ref=psdc_553398_t5_B00FZPHMUG</td></tr><tr><td>IWISS IWS-3220M Micro Connector Pin Crimping Tool 0.03-0.52mm&amp;sup2; 32-20AWG</td><td>Amazon</td><td>IWS-3220M</td><td>These are very common and there are many equivalents.&lt;br/&gt; https://www.amazon.com/gp/product/B078WPT5M1/ref=ppx_yo_dt_b_search_asin_title?ie=UTF8&amp;amp;psc=1</td></tr></tbody></table>

building a simple and versatile illumination system for optogenetic experiments

Controlling biological processes using light has increased the accuracy and speed with which researchers can manipulate many biological processes. Optical control allows for an unprecedented ability to dissect function and holds the potential for enabling novel genetic therapies. However, optogenetic experiments require adequate light sources with spatial, temporal, or intensity control, often a bottleneck for researchers. Here we detail how to build a low-cost and versatile LED illumination system that is easily customizable for different available optogenetic tools. This system is configurable for manual or computer control with adjustable LED intensity. We provide an illustrated step-by-step guide for building the circuit, making it computer-controlled, and constructing the LEDs. To facilitate the assembly of this device, we also discuss some basic soldering techniques and explain the circuitry used to control the LEDs. Using our open-source user interface, users can automate precise timing and pulsing of light on a personal computer (PC) or an inexpensive tablet. This automation makes the system useful for experiments that use LEDs to control genes, signaling pathways, and other cellular activities that span large time scales. For this protocol, no prior expertise in electronics is required to build all the parts needed or to use the illumination system to perform optogenetic experiments.

Watch this Scientific Journal Video about Building a Simple and Versatile Illumination System for Optogenetic Experiments at JoVE.com

Building a Simple and Versatile Illumination System for Optogenetic Experiments

This protocol describes how to perform optogenetic experiments for controlling gene expression with red and far-red light using PhyB and PIF3. Included are step-by-step instructions for building a simple and flexible illumination system, which enables the control of gene expression or other optogenetics with a computer.

Controlling biological processes using light has increased the accuracy and speed with which researchers can manipulate many biological processes. Optical ...

building-simple-versatile-illumination-system-for-optogenetic

Research

JoVE Journal

Bioengineering

3.7K Views.  University of California, San Diego. This protocol describes how to perform optogenetic experiments for controlling gene expression with red and far-red light using PhyB and PIF3. Included are step-by-step instructions for building a simple and flexible illumination system, which enables the control of gene expression or other optogenetics with a computer.

Video: Building a Simple and Versatile Illumination System for Optogenetic Experiments

Using Affordable LED Arrays for Photo-Stimulation of Neurons

Standard slice electrophysiology has allowed researchers to probe individual components of neural circuitry by recording electrical responses of single cells in response to electrical or pharmacological manipulations1,2. With the invention of methods to optically control genetically targeted neurons (optogenetics), researchers now have an unprecedented level of control over specific groups of neurons in the standard slice preparation. In particular, photosensitive channelrhodopsin-2 (ChR2) allows researchers to activate neurons with light3,4. By combining careful calibration of LED-based photostimulation of ChR2 with standard slice electrophysiology, we are able to probe with greater detail the role of adult-born interneurons in the olfactory bulb, the first central relay of the olfactory system. Using viral expression of ChR2-YFP specifically in adult-born neurons, we can selectively control young adult-born neurons in a milieu of older and mature neurons. Our optical control uses a simple and inexpensive LED system, and we show how this system can be calibrated to understand how much light is needed to evoke spiking activity in single neurons. Hence, brief flashes of blue light can remotely control the firing pattern of ChR2-transduced newborn cells.

Adult-born neurons expressing ChR2 can be manipulated in slice electrophysiological preparations in order to examine their contribution towards the function of olfactory neural circuits.

Standard slice electrophysiology has allowed researchers to probe individual components of neural circuitry by recording electrical responses of single ...

Neuroscience

Large Volume, Behaviorally-relevant Illumination for Optogenetics in Non-human Primates

This protocol describes a large-volume illuminator, which was developed for optogenetic manipulations in the non-human primate brain. The illuminator is a modified plastic optical fiber with etched tip, such that the light emitting surface area is &#62; 100x that of a conventional fiber. In addition to describing the construction of the large-volume illuminator, this protocol details the quality-control calibration used to ensure even light distribution. Further, this protocol describes techniques for inserting and removing the large volume illuminator. Both superficial and deep structures may be illuminated. This large volume illuminator does not need to be physically coupled to an electrode, and because the illuminator is made of plastic, not glass, it will simply bend in circumstances when traditional optical fibers would shatter. Because this illuminator delivers light over behaviorally-relevant tissue volumes (&#8776; 10 mm3) with no greater penetration damage than a conventional optical fiber, it facilitates behavioral studies using optogenetics in non-human primates.

A protocol to build a tissue penetrating illuminator for delivering light over large volumes with minimal diameter is presented.

This protocol describes a large-volume illuminator, which was developed for optogenetic manipulations in the non-human primate brain. The illuminator is a ...

Behavior

Bioluminescent Optogenetics 2.0: Harnessing Bioluminescence to Activate Photosensory Proteins In Vitro and In Vivo

Bioluminescence - light emitted by a luciferase enzyme oxidizing a small molecule substrate, a luciferin - has been used in vitro and in vivo to activate light-gated ion channels and pumps in neurons. While this bioluminescent optogenetics (BL-OG) approach confers a chemogenetic component to optogenetic tools, it is not limited to use in neuroscience. Rather, bioluminescence can be harnessed to activate any photosensory protein, thus enabling the manipulation of a multitude of light-mediated functions in cells. A variety of luciferase-luciferin pairs can be matched with photosensory proteins requiring different wavelengths of light and light intensities.
Depending on the specific application, efficient light delivery can be achieved by using luciferase-photoreceptor fusion proteins or by simple co-transfection. Photosensory proteins based on light-dependent dimerization or conformational changes can be driven by bioluminescence to effect cellular processes from protein localization, regulation of intracellular signaling pathways to transcription. The protocol below details the experimental execution of bioluminescence activation in cells and organisms and describes the results using bioluminescence-driven recombinases and transcription factors. The protocol provides investigators with the basic procedures for carrying out bioluminescent optogenetics in vitro and in vivo. The described approaches can be further extended and individualized to a multitude of different experimental paradigms.

Bioluminescent Optogenetics 2.0: Harnessing Bioluminescence to Activate Photosensory Proteins In Vitro and In Vivo

Bioluminescence-light emitted by a luciferase enzyme oxidizing a small-molecule substrate, a luciferin-can be harnessed to activate photosensory proteins, thereby adding another dimension to light stimulation and enabling the manipulation of a multitude of light-mediated functions in cells across temporal and spatial scales.

Bioluminescence - light emitted by a luciferase enzyme oxidizing a small molecule substrate, a luciferin - has been used in vitro and in vivo to activate ...

Biochemistry

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

Design and Implementation of an Automated Illuminating, Culturing, and Sampling System for Microbial Optogenetic Applications

Optogenetic systems utilize genetically-encoded proteins that change conformation in response to specific wavelengths of light to alter cellular processes. There is a need for culturing and measuring systems that incorporate programmed illumination and stimulation of optogenetic systems. We present a protocol for building and using a continuous culturing apparatus to illuminate microbial cells with programmed doses of light, and automatically acquire and analyze images of cells in the effluent. The operation of this apparatus as a chemostat allows the growth rate and the cellular environment to be tightly controlled. The effluent of the continuous cell culture is regularly sampled and the cells are imaged by multi-channel microscopy. The culturing, sampling, imaging, and image analysis are fully automated so that dynamic responses in the fluorescence intensity and cellular morphology of cells sampled from the culture effluent are measured over multiple days without user input. We demonstrate the utility of this culturing apparatus by dynamically inducing protein production in a strain of Saccharomyces cerevisiae engineered with an optogenetic system that activates transcription.

We designed a continuous culturing apparatus for use with optogenetic systems to illuminate cultures of microbes and regularly image cells in the effluent with an inverted microscope. The culturing, sampling, imaging, and image analysis are fully automated so that dynamic responses to illumination can be measured over several days.

Optogenetic systems utilize genetically-encoded proteins that change conformation in response to specific wavelengths of light to alter cellular ...

Investigating Development with Optogenetic Signaling Manipulation in Zebrafish

Optogenetic Signaling Activation in Zebrafish Embryos

Signaling pathways orchestrate fundamental biological processes, including development, regeneration, homeostasis, and disease. Methods to experimentally manipulate signaling are required to understand how signaling is interpreted in these wide-ranging contexts. Molecular optogenetic&#160;tools can provide reversible, tunable manipulations of signaling pathway activity with a high degree of spatiotemporal control and have been applied in vitro, ex vivo, and in vivo. These tools couple light-responsive protein domains, such as the blue light homodimerizing light-oxygen-voltage sensing (LOV) domain, with signaling effectors to confer light-dependent experimental control over signaling. This protocol provides practical guidelines for using the LOV-based bone morphogenetic protein (BMP) and Nodal signaling activators bOpto-BMP and bOpto-Nodal in the optically accessible early zebrafish embryo. It describes two control experiments: A quick phenotype assay to determine appropriate experimental conditions, and an immunofluorescence assay to directly assess signaling. Together, these control experiments can help establish a pipeline for using optogenetic tools in early zebrafish embryos. These strategies provide a powerful platform to investigate the roles of signaling in development, health, and physiology.

Author Spotlight: Manipulating Signaling in Zebrafish Embryos to Decode Cell Fate Decisions 

Optogenetic manipulation of signaling pathways can be a powerful strategy to investigate how signaling is decoded in development, regeneration, homeostasis, and disease. This protocol provides practical guidelines for using light-oxygen-voltage sensing domain-based Nodal and bone morphogenic protein (BMP) signaling activators in the early zebrafish embryo.

Signaling pathways orchestrate fundamental biological processes, including development, regeneration, homeostasis, and disease. Methods to experimentally ...

Developmental Biology

Reliably Engineering and Controlling Stable Optogenetic Gene Circuits in Mammalian Cells

Reliable gene expression control in mammalian cells requires tools with high fold change, low noise, and determined input-to-output transfer functions, regardless of the method used. Toward this goal, optogenetic gene expression systems have gained much attention over the past decade for spatiotemporal control of protein levels in mammalian cells. However, most existing circuits controlling light-induced gene expression vary in architecture, are expressed from plasmids, and utilize variable optogenetic equipment, creating a need to explore characterization and standardization of optogenetic components in stable cell lines. Here, the study provides an experimental pipeline of reliable gene circuit construction, integration, and characterization for controlling light-inducible gene expression in mammalian cells, using a negative feedback optogenetic circuit as a case example. The protocols also illustrate how standardizing optogenetic equipment and light regimes can reliably reveal gene circuit features such as gene expression noise and protein expression magnitude. Lastly, this paper may be of use for laboratories unfamiliar with optogenetics who wish to adopt such technology. The pipeline described here should apply for other optogenetic circuits in mammalian cells, allowing for more reliable, detailed characterization and control of gene expression at the transcriptional, proteomic, and ultimately phenotypic level in mammalian cells.

Reliably controlling light-responsive mammalian cells requires the standardization of optogenetic methods. Toward this goal, this study outlines a pipeline of gene circuit construction, cell engineering, optogenetic equipment operation, and verification assays to standardize the study of light-induced gene expression using a negative-feedback optogenetic gene circuit as a case study.

Reliable gene expression control in mammalian cells requires tools with high fold change, low noise, and determined input-to-output transfer functions, ...

Engineering and Characterization of an Optogenetic Model of the Human Neuromuscular Junction

Many neuromuscular diseases, such as myasthenia gravis (MG), are associated with dysfunction of the neuromuscular junction (NMJ), which is difficult to characterize in animal models due to physiological differences between animals and humans. Tissue engineering offers opportunities to provide in vitro models of functional human NMJs that can be used to diagnose and investigate NMJ pathologies and test potential therapeutics. By incorporating optogenetic proteins into induced pluripotent stem cells (iPSCs), we generated neurons that can be stimulated with specific wavelengths of light. If the NMJ is healthy and functional, a neurochemical signal from the motoneuron results in muscle contraction. Through the integration of optogenetics and microfabrication with tissue engineering, we established an unbiased and automated methodology for characterizing NMJ function using video analysis. A standardized protocol was developed for NMJ formation, optical stimulation with simultaneous video recording, and video analysis of tissue contractility. Stimulation of optogenetic motoneurons by light to induce skeletal muscle contractions recapitulates human NMJ physiology and allows for repeated functional measurements of NMJ over time and in response to various inputs. We demonstrate this platform&#39;s ability to show functional improvements in neuromuscular connectivity over time and characterize the damaging effects of patient MG antibodies or neurotoxins on NMJ function.

We describe a reproducible, automated, and unbiased imaging system for characterizing neuromuscular junction function using human engineered skeletal muscle tissue and optogenetic motoneurons. This system allows for the functional quantification of neuromuscular connectivity over time and detects diminished neuromuscular function caused by neurotoxins and myasthenia gravis patient serum.

Many neuromuscular diseases, such as myasthenia gravis (MG), are associated with dysfunction of the neuromuscular junction (NMJ), which is difficult to ...

Optrode Array for Simultaneous Optogenetic Modulation and Electrical Neural Recording

During the last decade, optogenetics has become an essential tool for the investigation of neural signaling due to its unique capability of selective neural modulation or monitoring. As specific types of neuronal cells can be genetically modified to express opsin proteins, optogenetics enables optical stimulation or inhibition of the selected neurons. There have been several technological advances in the optical system for optogenetics. Recently, it was proposed to combine the optical waveguide for light delivery with electrophysiological recording to simultaneously monitor the neural responses to optogenetic stimulation or inhibition. In this study, an implantable optrode array (2x2 optical fibers) was developed with embedded multichannel electrodes. 
A light-emitting diode (LED) was employed as a light source, and a microfabricated microlens array was integrated to provide sufficient light power at the tip of the optical fibers. The optrode array system comprises the disposable part and the reusable part. The disposable part has optical fibers and electrodes, while the reusable part has the LED and electronic circuitry for light control and neural signal processing. The novel design of the implantable optrode array system is introduced in the accompanying video in addition to the procedure of the optrode implantation surgery, optogenetic light stimulation, and the electrophysiological neural recording. The results of in vivo experiments successfully showed time-locked neural spikes evoked by the light stimuli from hippocampal excitatory neurons of mice.

Here, we present the fabrication method of an optrode system with optical fibers for light delivery and an electrode array for neural recording. In vivo experiments with transgenic mice expressing channelrhodopsin-2 show the feasibility of the system for simultaneous optogenetic stimulation and electrophysiological recording.

During the last decade, optogenetics has become an essential tool for the investigation of neural signaling due to its unique capability of selective ...

A Light-Stimulation Grid Approach to Study Cellular Optogenetic Protein

Mapping the Cellular Distribution of an Optogenetic Protein Using a Light-Stimulation Grid

Our goal was to accurately track the cellular distribution of an optogenetic protein and evaluate its functionality within a specific cytoplasmic location. To achieve this, we co-transfected cells with nuclear-targeted cAMP sensors and our laboratory-developed optogenetic protein, bacterial photoactivatable adenylyl cyclase-nanoluciferase (bPAC-nLuc). bPAC-nLuc, when stimulated with 445 nm light or luciferase substrates, generates adenosine 3&#39;,5&#39;-cyclic monophosphate (cAMP). We employed a solid-state laser illuminator connected to a point scanning system that allowed us to create a grid/matrix pattern of small illuminated spots (~1 &#181;m2) throughout the cytoplasm of HC-1 cells. By doing so, we were able to effectively track the distribution of nuclear-targeted bPAC-nLuc and generate a comprehensive cAMP response map. This map accurately represented the cellular distribution of bPAC-nLuc, and its response to light stimulation varied according to the amount of protein in the illuminated spot. This innovative approach contributes to the expanding toolkit of techniques available for investigating cellular optogenetic proteins. The ability to map its distribution and response with high precision has far-reaching potential and could advance various fields of research.

This protocol involves transfecting cAMP sensors and bPAC-nLuc, an optogenetic protein, to accurately track its cellular distribution and response to light stimulation. The innovative approach of creating a cAMP response map using a point scanning system holds the potential for advancing research with optogenetic proteins in different fields.

Building a Simple and Versatile Illumination System for Optogenetic Experiments

Summary

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Chapters in this video

Using Affordable LED Arrays for Photo-Stimulation of Neurons

Large Volume, Behaviorally-relevant Illumination for Optogenetics in Non-human Primates

Bioluminescent Optogenetics 2.0: Harnessing Bioluminescence to Activate Photosensory Proteins In Vitro and In Vivo

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Design and Implementation of an Automated Illuminating, Culturing, and Sampling System for Microbial Optogenetic Applications

Optogenetic Signaling Activation in Zebrafish Embryos

Reliably Engineering and Controlling Stable Optogenetic Gene Circuits in Mammalian Cells

Engineering and Characterization of an Optogenetic Model of the Human Neuromuscular Junction

Optrode Array for Simultaneous Optogenetic Modulation and Electrical Neural Recording

Mapping the Cellular Distribution of an Optogenetic Protein Using a Light-Stimulation Grid