The work was supported by EU through project FP7-PEOPLE-212-ITN 316832-OLIMPIA, 
Telethon - Italy (grants GGP12033 and GGP14022), Fondazione Cariplo (grant ID 2013-0738).

1. Preparation of Photoactive Substrates
<ol>
	<li>Prepare a P3HT:PCBM solution (1:1 w/w) in chlorobenzene at a P3HT concentration of 20 g/L. Mix the solution with a magnetic stirrer for at least 4 hr at 60 &#176;C. Consider a volume of 150 &#181;l of solution for each substrate to be prepared.</li>
	<li>Clean ITO-coated glass slides (Rs = 10 &#937;/sq, 18x18 mm2, thickness 170 &#181;m) with consecutive baths of deionized water, acetone and isopropanol in a sonicator (10 min for each step). Dry the coverslips with a nitrogen gun. Put the samples in a plasma cleaner at 100 W power for 10 min.</li>
	<li>Deposit a thin film of the active layer on the cleaned substrates via spin-coating.
	<ol>
		<li>With a diamond tip, cut a microscope glass slide, (thickness &#8776; 1 mm) to the same lateral dimensions of the ITO-coated substrates. Remove the P3HT:PCBM solution from the hotplate and let it cool to ambient temperature.</li>
		<li>Set a two-steps spin-coater program: i) 30 sec at 800 rpm; ii) 30 sec at 1,600 rpm. Place the slide on the chuck of the spin-coater and start the vacuum to fix it. Put a drop of acetone on the slide and then the cleaned substrate, with the ITO-coated side facing upwards. Start the spin-coater rotation to get rid of the excess acetone.</li>
		<li>Put 150 &#181;l of P3HT:PCBM solution on the substrates and start again the spin-coater (use the same protocol described in 1.3.2).</li>
		<li>After the spin-coater has finished, remove the sample and carefully detach the coated substrate from the glass slide. Clean the back of the substrate with a cleanroom swab dipped in acetone. 
		Note: The photoactive substrates can be stored for few days in a dark box until used. For longer periods, keep them in a glovebox with inert gas.</li>
	</ol>
	</li>
</ol>
2. Preparation of Cell Culture Medium and Electrophysiology Solutions
<ol>
	<li>Prepare the complete growth medium (CGM) for HEK-293 cells: Dulbecco Modified Eagle&#39;s Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS) not heat-inactivated, 100 U/ml penicillin and 100 &#181;g/ml streptomycin. Sterilize the medium with a 0.2 &#181;m filtration system and store at 4 &#176;C.</li>
	<li>Prepare the extracellular solution in ultrapure water (concentrations in mM): 135 NaCl, 5.4 KCl, 5 HEPES, 10 Glucose, 1.8 CaCl2, 1 MgCl2. Adjust pH to 7.4 with NaOH. Sterilize the solution with a 0.2 &#181;m filtration system and store at 4 &#176;C.</li>
	<li>Prepare the intracellular solution in ultrapure water (concentrations in mM): 12 KCl, 125 K-Gluconate, 1 MgCl2, 0.1 CaCl2, 10 EGTA, 10 HEPES, 10 ATP-Na2. Note that ATP-Na2 is heat-sensitive and add it last. Adjust pH to 7.4 with NaOH. Sterilize the solution with a 0.2 &#181;m filtration system, aliquot in 1 ml tubes and store at -20 &#176;C.</li>
</ol>
3. Plating HEK-293 Cells on the Active Substrates
<ol>
	<li>Place the photoactive substrate in a glass beaker covered with aluminum or a closed Petri dish and put in an oven at 120 &#176;C for 2 hr for sterilization. From this moment onwards, perform all operations in sterile conditions. Put the samples in a sterile hood and let them cool down.</li>
	<li>Coat the photoactive substrate with an adhesion layer to reduce the surface hydrophobicity and to promote cell adhesion.
	<ol>
		<li>Place the active substrates in a P35 Petri dish or a 6 multiwell plate. Given the hydrophobicity of the conjugated polymer layer, make a polydimethylsiloxane (PDMS) well to fix the sample and confine the solutions.
		<ol>
			<li>Mix PDMS and catalyst in a 10:1 proportion with a glass rod to obtain a viscous slurry.</li>
			<li>Pour the slurry in a 6 multiwell plate, dispensing an amount sufficient to cover half of each well.</li>
			<li>Wait for PDMS polymerization at RT.</li>
			<li>Cut the cured PDMS in the middle for obtaining a square shape of the same dimension of the polymer sample.</li>
			<li>Sterilize the PDMS wells by immersion in a 70% ethanol-water mixture for at least 30 min.</li>
		</ol>
		</li>
		<li>If using the PDMS well, scratch the photoactive layer along the entire border of the well with pointed tweezers, in order to avoid detachment of the entire layer when removing the PDMS well after cell culturing.</li>
		<li>Cover the entire surface of each sample with a fibronectin solution (2 mg/L in PBS). A volume between 500-750 &#181;l per sample is usually sufficient if a PDMS well is used (clear surface of the well: 15x15 mm2). Without the PDMS well, about 2 ml of fibronectin solution are necessary to completely immerse the sample; pay attention that the substrate does not float while adding the solution. Incubate the samples at 37 &#176;C for at least 1 hr.</li>
		<li>Remove the fibronectin solution with a glass pipette and wash once with 2 ml of PBS.</li>
	</ol>
	</li>
	<li>Plate HEK-293 cells on the fibronectin-treated samples at a density of 15,000 cells/cm2 in complete growth medium. Use a volume of 750-1,000 &#181;l of CGM for each sample if a PDMS well is used, otherwise 2-3 ml of CGM are necessary. Incubate the cells at 37 &#176;C and 5% CO2 for 24-48 hr.</li>
</ol>
4. Optical Stimulation Protocol and Electrophysiological Measurements
<ol>
	<li>Put the extracellular solution in a water bath at 37 &#176;C to thermalize; thaw the intracellular solution, put it in a 1 ml syringe with a 34 gauge needle and keep it in contact with ice to avoid degradation of the ATP.</li>
	<li>Take a cell-coated substrate from the incubator, carefully removing the PDMS well if present. Remove the growth medium with a glass pipette and rinse the sample with extracellular solution. Proceed slowly to avoid cell detachment.</li>
	<li>Put the device into the sample-holder of the electrophysiology station with extracellular solution and the reference electrode. Prepare fresh glass micropipettes for patch-clamp with a puller (ideal resistance is in the range 2-4 M&#937;). Fill half of the pipette with intracellular solution and mount it on the micromanipulator.</li>
	<li>Look for a healthy cell to patch on the device. To avoid unwanted excitation of the photoactive substrate, use IR illumination for imaging, placing a long-wavelength pass filter (for example, a cut wavelength &#955; = 750 nm can be used for polymers absorbing in the visible) in front of the microscope illuminator.</li>
	<li>Patch the selected cell and apply the desired protocol for optical stimulation by delivering light to the sample via the fluorescence excitation path of the microscope (Figure 1). A detailed description of the patch-clamp protocol can be found in reference 20.
	<ol>
		<li>Position the patch pipette in close proximity to the cell membrane with a micrometric manipulator. During the positioning, apply overpressure to the pipette in order to avoid dirt sticking on the tip. The pipette should be few microns above the cell.</li>
		<li>Start lowering the pipette towards the cell membrane while controlling the pipette resistance on the patch control software. When the pipette resistance has increased about 1 M&#8486;, remove the overpressure from the pipette while applying a gentle suction, in order to form the gigaseal between the pipette and the cell membrane (i.e., the measured resistance should increase at values greater than 1 G&#8486;).</li>
		<li>Apply a negative potential to the pipette close to the expected cell resting potential (for HEK293 cell a potential of -40 mV can be used) to help stabilize the seal. Compensate the capacitive transients due to the pipette capacitance with the relative commands on the patch amplifier and/or the patch control software.</li>
		<li>Break the membrane to allow electrical access to the cell cytoplasm by applying a brief and intense suction pulse to the pipette. Set the patch amplifier to current clamp mode (I = 0, i.e., with no current injected into the cell) to track the cell membrane potential. Wait few minutes to see if the cell potential is stable.</li>
		<li>Apply the desired illumination protocol to the cell and record the effect on the membrane potential. 
		Note: The illumination can be delivered to the sample either from the bottom or from the top, depending on the microscope architecture.
		<ol>
			<li>Select an excitation wavelength that is well absorbed by the active material; for the P3HT:PCBM blend, a wavelength in the 450-600 nm range can be used. Suitable photoexcitation densities are in the range 10-100 mW/mm2. 
			Note: Short pulses of light (below 10-20 msec) result in a transient depolarization of the cell, while with prolonged illumination (hundreds of milliseconds or longer pulses) a sustained hyperpolarization is observed until the light is switched off.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Alivisatos, A. P., 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=Nanotools+for+Neuroscience+and+Brain+Activity+Mapping.">Nanotools for Neuroscience and Brain Activity Mapping.</a> ACS Nano. 7 (3), 1850-1866 (2013).</li><li>Spira, M. E., Hai, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-electrode+array+technologies+for+neuroscience+and+cardiology.">Multi-electrode array technologies for neuroscience and cardiology.</a> Nat. Nanotechnol. 8 (2), 83-94 (2013).</li><li>Scanziani, M., H&#228;usser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiology+in+the+age+of+light.">Electrophysiology in the age of light.</a> Nature. 461 (7266), 930-939 (2009).</li><li>Bareket-Keren, L., Hanein, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+interfaces+for+light+directed+neuronal+stimulation:+advances+and+challenges.">Novel interfaces for light directed neuronal stimulation: advances and challenges.</a> Int. J. Nanomed. 9 (1), 65-83 (2014).</li><li>Antognazza, M. R., 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=Shedding+Light+on+Living+Cells.">Shedding Light on Living Cells.</a> Adv. Mater. , (2014).</li><li>Martino, N., Ghezzi, D., Benfenati, F., Lanzani, G., Antognazza, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organic+semiconductors+for+artificial+vision.">Organic semiconductors for artificial vision.</a> J. Mater. Chem. B. 1 (31), 3768-3780 (2013).</li><li>Antognazza, M. R., Scherf, U., Monti, P., Lanzani, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organic-based+tristimuli+colorimeter.">Organic-based tristimuli colorimeter.</a> Appl. Phys. Lett. 90 (16), 163509 (2007).</li><li>Liao, C., Zhang, M., Yao, M. Y., Hua, T., Li, L., Yan, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flexible+Organic+Electronics+in+Biology:+Materials+and+Devices.">Flexible Organic Electronics in Biology: Materials and Devices.</a> Adv. Mater. , (2014).</li><li>Khodagholy, 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=NeuroGrid:+recording+action+potentials+from+the+surface+of+the+brain.">NeuroGrid: recording action potentials from the surface of the brain.</a> Nat. Neurosci. 18 (2), 310-315 (2015).</li><li>Campana, 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=Electrocardiographic+Recording+with+Conformable+Organic+Electrochemical+Transistor+Fabricated+on+Resorbable+Bioscaffold.">Electrocardiographic Recording with Conformable Organic Electrochemical Transistor Fabricated on Resorbable Bioscaffold.</a> Adv. Mater. 26 (23), 3874-3878 (2014).</li><li>Bonetti, S., 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+Lysinated+Thiophene-Based+Semiconductor+as+a+Multifunctional+Neural+Bioorganic+Interface.">A Lysinated Thiophene-Based Semiconductor as a Multifunctional Neural Bioorganic Interface.</a> Adv. Healthc. Mater. , (2015).</li><li>Benfenati, V., 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+transparent+organic+transistor+structure+for+bidirectional+stimulation+and+recording+of+primary+neurons.">A transparent organic transistor structure for bidirectional stimulation and recording of primary neurons.</a> Nat. Mater. 12 (7), 672-680 (2013).</li><li>Rivnay, J., Owens, R. M., Malliaras, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Rise+of+Organic+Bioelectronics.">The Rise of Organic Bioelectronics.</a> Chem. Mater. 26 (1), 679-685 (2014).</li><li>Pires, F., 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=Neural+stem+cell+differentiation+by+electrical+stimulation+using+a+cross-linked+PEDOT+substrate:+Expanding+the+use+of+biocompatible+conjugated+conductive+polymers+for+neural+tissue+engineering.">Neural stem cell differentiation by electrical stimulation using a cross-linked PEDOT substrate: Expanding the use of biocompatible conjugated conductive polymers for neural tissue engineering.</a> BBA-Gen. Subjects. 1850 (6), 1158-1168 (2015).</li><li>Ghezzi, 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=A+hybrid+bioorganic+interface+for+neuronal+photoactivation.">A hybrid bioorganic interface for neuronal photoactivation.</a> Nat. Commun. 2 (166), 1-7 (2011).</li><li>Ghezzi, 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=A+polymer+optoelectronic+interface+restores+light+sensitivity+in+blind+rat+retinas.">A polymer optoelectronic interface restores light sensitivity in blind rat retinas.</a> Nat. Photonics. 7 (5), 400-406 (2013).</li><li>Benfenati, V., 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=Photostimulation+of+Whole-Cell+Conductance+in+Primary+Rat+Neocortical+Astrocytes+Mediated+by+Organic+Semiconducting+Thin+Films.">Photostimulation of Whole-Cell Conductance in Primary Rat Neocortical Astrocytes Mediated by Organic Semiconducting Thin Films.</a> Adv. Healthc. Mater. 3 (3), 392-399 (2014).</li><li>Martino, N., 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=Photothermal+cellular+stimulation+in+functional+bio-polymer+interfaces.">Photothermal cellular stimulation in functional bio-polymer interfaces.</a> Sci. Rep. 5 (8911), (2015).</li><li>Antognazza, M. R., Ghezzi, D., Musitelli, D., Garbugli, M., Lanzani, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+hybrid+solid-liquid+polymer+photodiode+for+the+bioenvironment.">A hybrid solid-liquid polymer photodiode for the bioenvironment.</a> Appl. Phys. Lett. 94 (24), 243501 (2009).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Essentials+of+Neuroscience.+Patch+Clamp+Electrophysiology.">Essentials of Neuroscience. Patch Clamp Electrophysiology.</a> JoVE Science Education Database. , (2015).</li><li>Scarpa, G., Idzko, A. L., G&#246;tz, S., Thalhammer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biocompatibility+Studies+of+Functionalized+Regioregular+Poly(3-hexylthiophene)+Layers+for+Sensing+Applications.">Biocompatibility Studies of Functionalized Regioregular Poly(3-hexylthiophene) Layers for Sensing Applications.</a> Macromol. Biosci. 10 (4), 378-383 (2010).</li><li>Bellani, S., 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=Reversible+P3HT/Oxygen+Charge+Transfer+Complex+Identification+in+Thin+Films+Exposed+to+Direct+Contact+with+Water.">Reversible P3HT/Oxygen Charge Transfer Complex Identification in Thin Films Exposed to Direct Contact with Water.</a> J. Phys. Chem. C. 118 (12), 6291-6299 (2014).</li><li>Wells, 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=Optical+stimulation+of+neural+tissue+in+vivo.">Optical stimulation of neural tissue in vivo.</a> Opt. Lett. 30 (5), 504-506 (2005).</li><li>Shapiro, M. G., Homma, K., Villarreal, S., Richter, C. P., Bezanilla, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infrared+light+excites+cells+by+changing+their+electrical+capacitance.">Infrared light excites cells by changing their electrical capacitance.</a> Nat. Commun. 3 (736), 1-10 (2012).</li><li>Duke, A. R., 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=Transient+and+selective+suppression+of+neural+activity+with+infrared+light.">Transient and selective suppression of neural activity with infrared light.</a> Sci. Rep. 3 (2600), 1-8 (2013).</li></ol>

The authors have nothing to disclose.

Critical steps of the reported protocol for in-vitro optical stimulation of cells mainly concern the choice of the light-sensitive polymer, the thermal sterilization parameters, the intensity and the duration of light stimuli. A P3HT:PCBM thin film was selected here, since it guarantees good temporal and electrochemical stability. However, one should notice that not all light-sensitive polymers can offer analogue performances,22 more specifically upon illumination. In addition, in this case selected t...

The possibility to manipulate the cellular activity with a precise spatial and temporal resolution represents a key strategy in neuroscientific research and in the treatment of neurological and psychiatric disorders.1 Traditional methods are based on electrical stimulation of cells using electrodes positioned in proximity or in contact with the targeted system,2 which can be of different complexity (single cell, cellular network, brain slices, in-vivo brain tissues). During the past century, the use of patch-clamp, metal and substrate-integrated electrodes have provided a detailed picture of the physiology and pathophysiology of single neurons and of the functioning mechanisms of neural networks. However, electrical stimulation suffers from important limitations. The first one is related to a generally poor spatial resolution due to the physical dimensions of the electrodes and their fixed geometry, which cannot be readily adapted to complex organized systems like biological tissues. Also, problems related to the electrodes impedance and of cross-talk between stimulation and recording systems may deteriorate the final signal-to-noise ratio of the measurements.3 On the other hand, the use of light for stimulation may help to overcome many limitations of the electrical approach. First of all, it offers unprecedented spatial (&#60; 1 &#181;m) and temporal resolution (&#60; 1 msec), making it possible to target specific cell types or even sub-cell compartments. In addition it is highly non-invasive since it avoids any physical contact with the tissue of interest and disentangles stimulation from recording. Moreover, both light intensity and wavelength can be precisely regulated and thus diverse stimulation protocols can be applied.3,4
However, the vast majority of animal cells do not present any specific sensitivity to light. Several strategies for optical stimulation have thus been proposed, either exploiting light-sensitive molecular mediators nearby or within the cells, or using photoactive device placed externally, close to the cell. The former category refers to endogenous mechanisms like the stimulation via visible or infrared (IR) light, as well as the use of either photoisomerizable/photocleavable compounds or the genetic expression of photosensitive molecular actuators (optogenetics). The latter class includes techniques for exogenous stimulation achieved with the use of inorganic nano/micro-particles or photoconductive silicon substrates.5 Nevertheless, all these systems have bright sides and drawbacks. In particular, endogenous absorption of cells in the visible range is weak and not reliable, and the concomitant generation of reactive oxygen species may be harmful to the cell. In general, IR is used for inducing local thermal heating due to water absorption, but the extinction coefficient of water is small, thus requiring strong infrared light (from tens to hundreds of W/mm2) that is difficult to deliver via standard microscope optics and may pose safety concerns for in-vivo applications. On the other hand, photo-switchable caging compounds have a time limited action and often require UV light that is hard to deliver due to limited tissue penetration. In addition they suffer from diffusion problems of the activated compounds upon photolysis outside the illuminated area. Finally, optogenetic tools have allowed scientists to target specific cellular subpopulation and sub-compartments and are rapidly emerging as one of the key technologies in neuroscientific research. However, inserting an exogenous DNA segment via a viral vector raises important safety issues, especially in view of adoption on human patients.5,6 For these reasons, research on new materials and devices capable of cell optical manipulation is an extremely hot topic.
Recently, a novel approach based on the use of light-sensitive conjugated polymers, able to efficiently transduce an optical stimulus into a modulation of cell electrical activity, has been proposed. The Cell Stimulation by Polymer Photoexcitation (CSPP) technique exploits many key-enabling features typical of organic semiconductors: they are intrinsically sensitive to light in the visible range;7 they are biocompatible, soft and conformable and their mechanical flexibility allows an intimate interface with tissue both in-vitro and in-vivo8-10. Apart from that, they can be easily functionalized to better adapt to the interface with living cells, and to enable specific excitation, probing and sensing capabilities.11,12 Moreover, they support electronic as well as ionic transport, making them ideal for the combination of electronics ad biology.13,14 Interestingly, they can work in photovoltaic mode, avoiding the need to apply an external bias for efficient cell optical stimulation.15 
The reliability of CSPP technique has been previously demonstrated in several systems, including primary neurons,15,16 astrocytes,17 secondary cell lines18 and explanted retinal tissues.16 In this work, all the steps necessary to fabricate a light-sensitive bio-polymer interface19 for optical stimulation of in-vitro systems are described in detail. As a study case, a prototypical organic photovoltaic blend of region-regular poly(3-hexylthiophene) (rr-P3HT), functioning as the electron donor, and phenyl-C61-butyric-acid-methyl ester (PCBM), acting as the electron acceptor is employed. As the biological system, Human Embryonic Kidney (HEK-293) cells are used. An example of a photostimulation protocol with the relative recording of cell activity via electrophysiological measurements is provided.
The described platform is however of general validity, and it can be easily extended to the use of other conjugated polymers (by properly adjusting the solution preparation process and the deposition parameters), different cell types (by properly changing the cell culture protocol, plating procedure and time requested for cell seeding and proliferation) and different stimulation protocols (light wavelength, stimuli frequency and duration, photoexcitation density).

<table border="0" cellpadding="0" cellspacing="0" width="693">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">rr-P3HT</td>
			<td style="width:120px;">Sigma Aldrich</td>
			<td style="width:191px;">698989-5G</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ITO-coated substrates</td>
			<td style="width:120px;">Nano-CS</td>
			<td style="width:191px;">IT10300100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fibronectin</td>
			<td style="width:120px;">Sigma Aldrich</td>
			<td style="width:191px;">F1141</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">chlorobenzene</td>
			<td style="width:120px;">Sigma Aldrich</td>
			<td align="right" style="width:191px;">319996</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PCBM</td>
			<td style="width:120px;">Nano-C</td>
			<td style="width:191px;">Nano-CPCBM-BF</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">acetone&#160;</td>
			<td style="width:120px;">Sigma Aldrich</td>
			<td align="right" style="width:191px;">270725</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">isopropyl alcohol</td>
			<td style="width:120px;">Sigma Aldrich</td>
			<td align="right" style="width:191px;">563935</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">HEK cells</td>
			<td style="width:120px;">LGC standards srl</td>
			<td style="width:191px;">ATCC-CRL-1573</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HEPES</td>
			<td style="width:120px;">Sigma Aldrich</td>
			<td style="width:191px;">H0887</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PBS</td>
			<td style="width:120px;">Sigma Aldrich</td>
			<td style="width:191px;">P5244</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">E-MEM</td>
			<td style="width:120px;">LGC standards srl</td>
			<td style="width:191px;">ATCC-30-2003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EDTA</td>
			<td style="width:120px;">Sigma Aldrich</td>
			<td style="width:191px;">E8008-</td>
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			<td height="42" style="height:42px;width:216px;">FBS</td>
			<td style="width:120px;">LGC standards srl</td>
			<td style="width:191px;">ATCC-30-2020</td>
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optical control of living cells electrical activity by conjugated polymers

Hybrid interfaces between organic semiconductors and living tissues represent a new tool for in-vitro and in-vivo applications. In particular, conjugated polymers display several optimal properties as substrates for biological systems, such as good biocompatibility, excellent mechanical properties, cheap and easy processing technology, and possibility of deposition on light, thin and flexible substrates. These materials have been employed for cellular interfaces like neural probes, transistors for excitation and recording of neural activity, biosensors and actuators for drug release. Recent experiments have also demonstrated the possibility to use conjugated polymers for all-optical modulation of the electrical activity of cells. Several in-vitro study cases have been reported, including primary neuronal networks, astrocytes and secondary line cells. Moreover, signal photo-transduction mediated by organic polymers has been shown to restore light sensitivity in degenerated retinas, suggesting that these devices may be used for artificial retinal prosthesis in the future. All in all, light sensitive conjugated polymers represent a new approach for optical modulation of cellular activity.
In this work, all the steps required to fabricate a bio-polymer interface for optical excitation of living cells are described. The function of the active interface is to transduce the light stimulus into a modulation of the cell membrane potential. As a study case, useful for in-vitro studies, a polythiophene thin film is used as the functional, light absorbing layer, and Human Embryonic Kidney (HEK-293) cells are employed as the biological component of the interface. Practical examples of successful control of the cell membrane potential upon stimulation with light pulses of different duration are provided. In particular, it is shown that both depolarizing and hyperpolarizing effects on the cell membrane can be achieved depending on the duration of the light stimulus. The reported protocol is of general validity and can be straightforwardly extended to other biological preparations.

Cells can be easily cultured on P3HT:PCBM substrates, provided that a suitable adhesion layer is deposited (like the fibronectin used in step 3.2 of the described protocol). P3HT:PCBM optical absorption peaks in the green part of the visible spectrum; however other light-sensitive conjugated polymers can be selected, according to the preferred photostimulation wavelengths range (Figure 2). Biocompatibility of these substrates has been demonstrated not only with cell lines...

Watch this Scientific Journal Video about Optical Control of Living Cells Electrical Activity by Conjugated Polymers at JoVE.com

Optical Control of Living Cells Electrical Activity by Conjugated Polymers

A method for stimulation of in-vitro cell cultures electrical activity with visible light, based on the use of organic semiconducting polymers is described.

Hybrid interfaces between organic semiconductors and living tissues represent a new tool for in-vitro and in-vivo applications. In particular, conjugated ...

optical-control-living-cells-electrical-activity-conjugated

Research

JoVE Journal

Bioengineering

7.5K Views.  Istituto Italiano di Tecnologia. A method for stimulation of in-vitro cell cultures electrical activity with visible light, based on the use of organic semiconducting polymers is described.

Video: Optical Control of Living Cells Electrical Activity by Conjugated Polymers

Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells

Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular events. Viscosity is one of the key parameters affecting the diffusion of molecules and proteins, and changes in viscosity have been linked to disease and malfunction at the cellular level.1-3 While methods to measure the bulk viscosity are well developed, imaging microviscosity remains a challenge. Viscosity maps of microscopic objects, such as single cells, have until recently been hard to obtain. Mapping viscosity with fluorescence techniques is advantageous because, similar to other optical techniques, it is minimally invasive, non-destructive and can be applied to living cells and tissues.
Fluorescent molecular rotors exhibit fluorescence lifetimes and quantum yields which are a function of the viscosity of their microenvironment.4,5 Intramolecular twisting or rotation leads to non-radiative decay from the excited state back to the ground state. A viscous environment slows this rotation or twisting, restricting access to this non-radiative decay pathway. This leads to an increase in the fluorescence quantum yield and the fluorescence lifetime. Fluorescence Lifetime Imaging (FLIM) of modified hydrophobic BODIPY dyes that act as fluorescent molecular rotors show that the fluorescence lifetime of these probes is a function of the microviscosity of their environment.6-8 A logarithmic plot of the fluorescence lifetime versus the solvent viscosity yields a straight line that obeys the F&ouml;rster Hoffman equation.9 This plot also serves as a calibration graph to convert fluorescence lifetime into viscosity.
Following incubation of living cells with the modified BODIPY fluorescent molecular rotor, a punctate dye distribution is observed in the fluorescence images. The viscosity value obtained in the puncta in live cells is around 100 times higher than that of water and of cellular cytoplasm.6,7 Time-resolved fluorescence anisotropy measurements yield rotational correlation times in agreement with these large microviscosity values. Mapping the fluorescence lifetime is independent of the fluorescence intensity, and thus allows the separation of probe concentration and viscosity effects. 
In summary, we have developed a practical and versatile approach to map the microviscosity in cells based on FLIM of fluorescent molecular rotors.

Fluorescence Lifetime Imaging (FLIM) has emerged as a key technique to image the environment and interaction of specific proteins and dyes in living cells. FLIM of fluorescent molecular rotors allows mapping of viscosity in living cells.

Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular ...

Spatio-Temporal Manipulation of Small GTPase Activity at Subcellular Level and on Timescale of Seconds in Living Cells

Dynamic regulation of the Rho family of small guanosine triphosphatases (GTPases) with great spatiotemporal precision is essential for various cellular functions and events1, 2. Their spatiotemporally dynamic nature has been revealed by visualization of their activity and localization in real time3. In order to gain deeper understanding of their roles in diverse cellular functions at the molecular level, the next step should be perturbation of protein activities at a precise subcellular location and timing. 
To achieve this goal, we have developed a method for light-induced, spatio-temporally controlled activation of small GTPases by combining two techniques: (1) rapamycin-induced FKBP-FRB heterodimerization and (2) a photo-caging method of rapamycin. With the use of rapamycin-mediated FKBP-FRB heterodimerization, we have developed a method for rapidly inducible activation or inactivation of small GTPases including Rac4, Cdc424, RhoA4 and Ras5, in which rapamycin induces translocation of FKBP-fused GTPases, or their activators, to the plasma membrane where FRB is anchored. For coupling with this heterodimerization system, we have also developed a photo-caging system of rapamycin analogs. A photo-caged compound is a small molecule whose activity is suppressed with a photocleavable protecting group known as a caging group. To suppress heterodimerization activity completely, we designed a caged rapamycin that is tethered to a macromolecule such that the resulting large complex cannot cross the plasma membrane, leading to virtually no background activity as a chemical dimerizer inside cells6. Figure 1 illustrates a scheme of our system. With the combination of these two systems, we locally recruited a Rac activator to the plasma membrane on a timescale of seconds and achieved light-induced Rac activation at the subcellular level6.

A method for spatio-temporal control of small GTPase activity by light is described. This method is based on rapamycin-induced FKBP-FRB heterodimerization and photo-caging systems. Optimization of light-irradiation enables the spatio-temporally controlled activation of small GTPases at the subcellular level.

Dynamic regulation of the Rho family of small guanosine triphosphatases (GTPases) with great spatiotemporal precision is essential for various cellular ...

Measuring the Mechanical Properties of Living Cells Using Atomic Force Microscopy

Mechanical properties of cells and extracellular matrix (ECM) play important roles in many biological processes including stem cell differentiation, tumor formation, and wound healing. Changes in stiffness of cells and ECM are often signs of changes in cell physiology or diseases in tissues. Hence, cell stiffness is an index to evaluate the status of cell cultures. Among the multitude of methods applied to measure the stiffness of cells and tissues, micro-indentation using an Atomic Force Microscope (AFM) provides a way to reliably measure the stiffness of living cells. This method has been widely applied to characterize the micro-scale stiffness for a variety of materials ranging from metal surfaces to soft biological tissues and cells. The basic principle of this method is to indent a cell with an AFM tip of selected geometry and measure the applied force from the bending of the AFM cantilever. Fitting the force-indentation curve to the Hertz model for the corresponding tip geometry can give quantitative measurements of material stiffness. This paper demonstrates the procedure to characterize the stiffness of living cells using AFM. Key steps including the process of AFM calibration, force-curve acquisition, and data analysis using a MATLAB routine are demonstrated. Limitations of this method are also discussed.

This paper demonstrates a protocol to characterize the mechanical properties of living cells by means of microindentation using an Atomic Force Microscope (AFM).

Mechanical  properties of cells and extracellular matrix (ECM) play important roles in many  biological processes including stem cell differentiation, ...

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as a model microorganism. The biosensor relies on direct binding of the target bacteria cells onto its surface, while no pretreatment (e.g.&#160;by cell lysis) of the studied sample is required. A mesoporous Si thin film is used as the optical transducer element of the biosensor. Under white light illumination, the porous layer displays well-resolved Fabry-P&#233;rot fringe patterns in its reflectivity spectrum. Applying a fast Fourier transform (FFT) to reflectivity data results in a single peak. Changes in the intensity of the FFT peak are monitored. Thus, target bacteria capture onto the biosensor surface, through antibody-antigen interactions, induces measurable changes in the intensity of the FFT peaks, allowing for a &#39;real time&#39; observation of bacteria attachment.
The mesoporous Si film, fabricated by an electrochemical anodization process, is conjugated with monoclonal antibodies, specific to the target bacteria. The immobilization, immunoactivity and specificity of the antibodies are confirmed by fluorescent labeling experiments. Once the biosensor is exposed to the target bacteria, the cells are directly captured onto the antibody-modified porous Si surface. These specific capturing events result in intensity changes in the thin-film optical interference spectrum of the biosensor. We demonstrate that these biosensors can detect relatively low bacteria concentrations (detection limit of 104 cells/ml) in less than an hour.

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

A label-free optical biosensor for rapid bacteria detection is introduced. The biosensor is based on a nanostructured porous Si, which is designed to directly capture the target bacteria cells onto its surface. We use monoclonal antibodies, immobilized onto the porous transducer, as the capture probes. Our studies demonstrate the applicability of these biosensors for the detection of low bacterial concentrations within minutes with no prior sample processing (such as cell lysis).

A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as a ...

Nanomanipulation of Single RNA Molecules by Optical Tweezers

A large portion of the human genome is transcribed but not translated. In this post genomic era, regulatory functions of RNA have been shown to be increasingly important. As RNA function often depends on its ability to adopt alternative structures, it is difficult to predict RNA three-dimensional structures directly from sequence. Single-molecule approaches show potentials to solve the problem of RNA structural polymorphism by monitoring molecular structures one molecule at a time. This work presents a method to precisely manipulate the folding and structure of single RNA molecules using optical tweezers. First, methods to synthesize molecules suitable for single-molecule mechanical work are described. Next, various calibration procedures to ensure the proper operations of the optical tweezers are discussed. Next, various experiments are explained. To demonstrate the utility of the technique, results of mechanically unfolding RNA hairpins and a single RNA kissing complex are used as evidence. In these examples, the nanomanipulation technique was used to study folding of each structural domain, including secondary and tertiary, independently. Lastly, the limitations and future applications of the method are discussed.

Optical tweezers have been used to study RNA folding by stretching individual molecules from their 5&#8217; and 3&#8217; ends. Here common procedures are described to synthesize RNA molecules for tweezing, calibration of the instrument, and methods to manipulate single molecules.

A large portion of the human genome is transcribed but not translated. In this post genomic era, regulatory functions of RNA have been shown to be ...

Transmission of Multiple Signals through an Optical Fiber Using Wavefront Shaping

The transmission of multiple independent optical signals through a multimode fiber is accomplished using wavefront shaping in order to compensate for the light distortion during the propagation within the fiber. Our methodology is based on digital optical phase conjugation employing only a single spatial light modulator, where the optical wavefront is individually modulated at different regions of the modulator, one region per light signal. Digital optical phase conjugation approaches are considered to be faster than other wavefront shaping approaches, where (for example) a complete determination of the wave propagation behavior of the fiber is performed. In contrast, the presented approach is time-efficient since it only requires one calibration per light signal. The proposed method is potentially appropriate for spatial division multiplexing in communications engineering. Further application fields are endoscopic light delivery in biophotonics, especially in optogenetics, where single cells in biological tissue have to be selectively illuminated with high spatial and temporal resolution.

We demonstrate the transmission of multiple independent signals through a multimode fiber using wavefront shaping employing a single spatial light modulator. By modulating the wavefront for each signal individually, spatially separated foci are transmitted. Potential applications are multiplexed data transfer in communications engineering and endoscopic light delivery in biophotonics.

The transmission of multiple independent optical signals through a multimode fiber is accomplished using wavefront shaping in order to compensate for the ...

Trapping of Micro Particles in Nanoplasmonic Optical Lattice

The plasmonic optical tweezer has been developed to overcome the diffraction limits of the conventional far field optical tweezer. Plasmonic optical lattice consists of an array of nanostructures, which exhibit a variety of trapping and transport behaviors. We report the experimental procedures to trap micro-particles in a simple square nanoplasmonic optical lattice. We also describe the optical setup and the nanofabrication of a nanoplasmonic array. The optical potential is created by illuminating an array of gold nanodiscs with a Gaussian beam of 980 nm wavelength, and exciting plasmon resonance. The motion of particles is monitored by fluorescence imaging. A scheme to suppress photothermal convection is also described to increase usable optical power for optimal trapping. Suppression of convection is achieved by cooling the sample to a low temperature, and utilizing the near-zero thermal expansion coefficient of a water medium. Both single particle transport and multiple particle trapping are reported here.

We describe a procedure to optically trap micro-particles in nanoplasmonic optical lattice.

The plasmonic optical tweezer has been developed to overcome the diffraction limits of the conventional far field optical tweezer. Plasmonic optical ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Silicon Nanowires and Optical Stimulation for Investigations of Intra- and Intercellular Electrical Coupling

Myofibroblasts can spontaneously internalize silicon nanowires (SiNWs), making them an attractive target for bioelectronic applications. These cell-silicon hybrids offer leadless optical modulation capabilities with minimal perturbation to normal cell behavior. The optical capabilities are obtained by the photothermal and photoelectric properties of SiNWs. These hybrids can be harvested using standard tissue culture techniques and then applied to different biological scenarios. We demonstrate here how these hybrids can be used to study the electrical coupling of cardiac cells and compare how myofibroblasts couple to one another or to cardiomyocytes. This process can be accomplished without special equipment beyond a fluorescent microscope with coupled laser line. Also shown is the use of a custom-built MATLAB routine that allows the quantification of calcium propagation within and between the different cells in the culture. Myofibroblasts are shown to have a slower electrical response than that of cardiomyocytes. Moreover, the myofibroblast intercellular propagation shows slightly slower, though comparable velocities to their intracellular velocities, suggesting passive propagation through gap junctions or nanotubes. This technique is highly adaptable and can be easily applied to other cellular arenas, for in vitro as well as in vivo or ex vivo investigations.

This protocol describes the use of silicon nanowires for intracellular optical bio-modulation of cell in a simple and easy to perform method. The technique is highly adaptable to diverse cell types and can be used for in vitro as well as in vivo applications.

Myofibroblasts can spontaneously internalize silicon nanowires (SiNWs), making them an attractive target for bioelectronic applications. These ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

Optical Control of Living Cells Electrical Activity by Conjugated Polymers

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Rozdziały w tym wideo

Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells

Spatio-Temporal Manipulation of Small GTPase Activity at Subcellular Level and on Timescale of Seconds in Living Cells

Measuring the Mechanical Properties of Living Cells Using Atomic Force Microscopy

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

Nanomanipulation of Single RNA Molecules by Optical Tweezers

Transmission of Multiple Signals through an Optical Fiber Using Wavefront Shaping

Trapping of Micro Particles in Nanoplasmonic Optical Lattice

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

Silicon Nanowires and Optical Stimulation for Investigations of Intra- and Intercellular Electrical Coupling

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes