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>

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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 thermal sterilization parameters do not lead to sizable degradation of the polymer optoelectronic features; notice however that, in case the sterilization method or the thermal treatment parameters are changed, possible effects on the polymer properties should be carefully evaluated. It is known, for instance, that UV exposure rapidly leads to irreversible polymer bleaching and degradation, due to conjugation loss. Furthermore, different annealing temperatures and duration may lead to different morphological order of the polymer surface, with negative effects on the electrical charge transport properties. The parameters of the photoexcitation protocol should be carefully evaluated as well: excitation densities above 100 mW/cm2, and/or prolonged stimuli, may easily lead to polymer degradation and failure of the photostimulation protocol. Another critical step in the protocol consists in the use of a protein adhesion layer, due to the high hydrophobicity degree of the P3HT:PCBM surface. If no adhesion layer is employed in the realization of the bio-polymer interface, cell seeding and proliferation might be seriously compromised.
Within the above mentioned critical steps and constraints, however, the reported protocol can be straightforwardly modified, according to the specific experimental needs and objectives. Other stable, light-sensitive polymers can be found,7 from several commercial suppliers or by properly synthesizing new compounds. This allows the use of different excitation wavelengths, spanning from the blue to the red and NIR range. Moreover, the polymer deposition technique is not restricted to the spin coating use. Indeed, other methods ensuring good homogeneity and suitable film thickness can be envisaged, such as ink-jet printing, spray coating, or blading The effects of the prolonged contact with the cell growing media and of the sterilization method should be evaluated for each new material taken into consideration. Even though the use of protein adhesion layers is necessary to obtain good cell cultures on top of the polymer surfaces, the specific use of fibronectin, as reported here, is not mandatory. Different adhesion protein layers may be used, also in accordance with the cell line to grow;21 for example, neuronal cells are usually cultured on a poly-L-lysine layer. Finally, illumination protocols can be changed and adapted to specific needs, in terms of wavelength, spot size, power excitation density, time stimuli duration and frequency, light incidence direction (from the substrate or from the electrolyte bath). As briefly stated above, however, the definition of the protocol parameters should take into account the polymer optical absorption range and possible degradation effects, which may vary a lot from case to case.
The limited temporal and electrochemical stability of some light sensitive polymers actually represents the main limitations of the technique. In addition, use of commercial polymers without any further purification may lead in some cases to limited repeatability of results among different batches of the material, due to different purity degree.
We also notice that the precise mechanisms at the base of the phototransduction effect in CSPP protocols still need to be further elucidated and characterized. Thermal, electrical and chemical phenomena are possibly involved, at different extents in different biological models. A complete understanding will be key to fully exploit the technique. In particular, the occurrence of heat-mediated phenomena should be reviewed as a limitation of the technique, since it can be hardly controlled, and can lead to local overheating effects, in particular in the case of in vivo applications. A specific implementation of the device, aimed at controlling the heat diffusion in the extracellular environment by proper engineering of heat-conductive paths, might reveal necessary in the next future for the full exploitation of the technique.
In literature, different techniques that use optical pulses for temperature-mediated stimulation of cells and tissues, both in-vitro and in-vivo can be found. In these studies, the absorption of IR laser beams by water is usually used as a transduction mechanism.23-25 With respect to Infrared Neural Stimulation, the CSPP mechanism has distinct advantages, especially for in-vitro experimentation. CSPP is based on light in the visible range and of moderate intensity, so that stimulation can be provided by the excitation path of a standard fluorescence microscope. On the contrary, water absorption requires wavelengths in the IR (mainly around 1.45 &#181;m and 1.93 &#181;m), which have to be delivered to the sample via external sources like optical fibers micromanipulated in the proximity of the preparation, since the optical train of a standard microscope cannot be used at such wavelengths. In the case of CSPP, the light patterns obtained with laser scanning systems as well as the spatial light modulators can be directly coupled to the optical train of most microscopes. In this way, the photostimulation of substrates based on organic semiconductors can make it possible to obtain stimulation of several cells in the field of view independently, with both high temporal and spatial resolution.
Another highly valuable alternative for optical stimulation is offered by genetic methods, based on targetable expression of genetically photoactivatable probes in cells. Optogenetics offers nowadays unprecedented temporal and spatial resolution, with typical illumination intensities in the range of few tens of mW/mm2, can both excite and inhibit electrical activity, and can be genetically targeted to specific sub-populations of specific brain regions of interest. However, it still presents some issues that have to be solved. In particular, safety concerns regarding the use of viruses for gene expression, especially in humans, and the achievement of stable and controlled long-term heterologous protein expression still represent the major challenges. CSPP technique allows to circumvent the need for gene transfer, and all related safety concerns, which are particularly relevant for in-vivo applications.
All in all, the CSPP method offers several advantages with respect to endogenous photostimulation techniques. Since it is not invasive, it can be easily coupled to any existing electrophysiological working station and it does not require complex laser sources or genetic transfection, while maintaining high spatial and temporal selectivity. For these reasons, CSPP holds promise to become a complementary tool in neuroscience in-vitro investigations, and to open new perspectives in in-vivo applications. However, a big effort from the material science, physics and engineering communities is expected in the forthcoming years, needed to refine, optimize and fully exploit the technique.

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><tbody><tr><td>rr-P3HT</td><td>Sigma Aldrich</td><td>698989-5G</td><td></td></tr><tr><td>ITO-coated substrates</td><td>Nano-CS</td><td>IT10300100</td><td></td></tr><tr><td>Fibronectin</td><td>Sigma Aldrich</td><td>F1141</td><td></td></tr><tr><td>chlorobenzene</td><td>Sigma Aldrich</td><td>319996</td><td></td></tr><tr><td>PCBM</td><td>Nano-C</td><td>Nano-CPCBM-BF</td><td></td></tr><tr><td>acetone&amp;nbsp;</td><td>Sigma Aldrich</td><td>270725</td><td></td></tr><tr><td>isopropyl alcohol</td><td>Sigma Aldrich</td><td>563935</td><td></td></tr><tr><td>HEK cells</td><td>LGC standards srl</td><td>ATCC-CRL-1573</td><td></td></tr><tr><td>HEPES</td><td>Sigma Aldrich</td><td>H0887</td><td></td></tr><tr><td>PBS</td><td>Sigma Aldrich</td><td>P5244</td><td></td></tr><tr><td>E-MEM</td><td>LGC standards srl</td><td>ATCC-30-2003</td><td></td></tr><tr><td>EDTA</td><td>Sigma Aldrich</td><td>E8008-</td><td></td></tr><tr><td>FBS</td><td>LGC standards srl</td><td>ATCC-30-2020</td><td></td></tr></tbody></table>

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 lines18,21 like HEK-293, but also with primary cultures of neurons15 and astrocytes.17 A typical micrograph of healthy HEK-293 cells cultured on a P3HT:PCBM film is shown in Figure 3. Optical stimulation of cells mediated by the photoactive substrates can result in different effects on the cell membrane, depending on the duration of the light stimulus.18 Upon the onset of the light pulse, a fast spike (with an intensity of about 3 mV and a duration of about 1 msec) can be observed in the cell membrane potential recordings (Figure 4). This signal is due to a capacitive charging of the polymer/electrolyte interface upon charge generation in the active material.
After this initial fast spiking, a transient depolarization (with an intensity of about 1 mV and a duration on the order of tens of milliseconds) is observed in the cell (Figure 4). For short pulses of light, as the light is switched off, an opposite behavior is observed. These signals have been attributed to a variation in membrane capacitance due to local heating following light absorption.
For prolonged illumination, however, the initial depolarization during the light pulses turns into a cell hyperpolarization (Figure 5). This phenomenon has again a thermal origin, but in this case it is related to a variation of the membrane equilibrium potential due to a change in the ion channels conductivities induced by the increased temperature.
<img alt="Figure 1" src="/files/ftp_upload/53494/53494fig1.jpg" /> 
Figure 1. Sketch of the photoactive substrate. The photoactive interfaces used for cellular optical stimulation are made of a thin film of organic semiconductors deposited on a ITO-coated glass substrate. Cells can be grown directly on the device surfaces after it has been treated with a suitable adhesion layer (e.g., fibronectin). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53494/53494fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53494/53494fig2.jpg" /> 
Figure 2. Absorption spectra of different conjugated polymers. The use of thin films of organic semiconductors may allow great tunability in excitation wavelength while retaining a partial transparence of the substrate. As an example, the absorption spectra of different conjugated polymers typically used in photovoltaics are reported. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53494/53494fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53494/53494fig3.jpg" /> 
Figure 3. HEK-293 cells cultured on a photoactive substrate. Typical micrograph of HEK-293 cells cultured on the surface of a photoactive substrate after 24 hr of incubation. Scale bar, 20 &#956;m. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53494/53494fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/53494/53494fig4.jpg" /> 
Figure 4. Optical stimulation of HEK-293 cells with 20 msec light pulses. Variation of the membrane potential of an HEK-293 cell elicited by a 20 msec pulse of light. An initial fast spike upon the onset of the light is followed by a depolarization of the cell during the pulse. As the light is switched off, an opposite behavior is observed. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53494/53494fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/53494/53494fig5.jpg" /> 
Figure 5. Optical stimulation of HEK-293 cells with 200 msec light pulses. Variation in the membrane potential of an HEK-293 cell elicited by a 200 msec pulse of light. The initial depolarization observed for short illumination is only a transient phenomenon, which after few tens of milliseconds of prolonged illumination turns into a hyperpolarization of the cell. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53494/53494fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

Universidad San Sebastian

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

An Optogenetic Method to Control and Analyze Gene Expression Patterns in Cell-to-cell Interactions

Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling pathway is one of such essential molecular machinery for cell-to-cell communications, which plays key roles in normal development of embryos. This pathway involves a cell-to-cell transfer of oscillatory information with ultradian rhythms, but despite the progress in molecular biology techniques, it has been challenging to elucidate the impact of multicellular interactions on oscillatory gene dynamics. Here, we present a protocol that permits optogenetic control and live monitoring of gene expression patterns in a precise temporal manner. This method successfully revealed that intracellular and intercellular periodic inputs of Notch signaling entrain intrinsic oscillations by frequency tuning and phase shifting at the single-cell resolution. This approach is applicable to the analysis of the dynamic features of various signaling pathways, providing a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems.

Here, we present a protocol to analyze cell-to-cell transfer of oscillatory information by optogenetic control and live monitoring of gene expression. This approach provides a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems.

Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling ...

Genetics

Monitoring the Effects of Illumination on the Structure of Conjugated Polymer Gels Using Neutron Scattering

We demonstrate a protocol to effectively monitor the gelation process of a high concentration solution of conjugated polymer both in the presence and absence of white light exposure. By instituting a controlled temperature ramp, the gelation of these materials can be precisely monitored as they proceed through this structural evolution, which effectively mirrors the conditions experienced during the solution deposition phase of organic electronic device fabrication. Using small angle neutron scattering (SANS) and ultra-small angle neutron scattering (USANS) along with appropriate fitting protocols we quantify the evolution of select structural parameters throughout this process. Thorough analysis indicates that continued light exposure throughout the gelation process significantly alters the structure of the ultimately formed gel. Specifically, the aggregation process of poly(3-hexylthiophene-2,5-diyl) (P3HT) nano-scale aggregates is negatively affected by the presence of illumination, ultimately resulting in the retardation of growth in conjugated polymer microstructures and the formation of smaller scale macro-aggregate clusters.

A protocol for the analysis of gels formed from the optoelectronic conjugated polymer poly(3-hexylthiophene-2,5-diyl) (P3HT) using small and ultra-small angle neutron scattering in both the presence and absence of illumination is presented.

We demonstrate a protocol to effectively monitor the gelation process of a high concentration solution of conjugated polymer both in the presence and ...

Chemistry

Fabrication of Polymer Microspheres for Optical Resonator and Laser Applications

This paper describes three methods of preparing fluorescent microspheres comprising &#960;-conjugated or non-conjugated polymers: vapor diffusion, interface precipitation, and mini-emulsion. In all methods, well-defined, micrometer-sized spheres are obtained from a self-assembling process in solution. The vapor diffusion method can result in spheres with the highest sphericity and surface smoothness, yet the types of the polymers able to form these spheres are limited. On the other hand, in the mini-emulsion method, microspheres can be made from various types of polymers, even from highly crystalline polymers with coplanar, &#960;-conjugated backbones. The photoluminescent (PL) properties from single isolated microspheres are unusual: the PL is confined inside the spheres, propagates at the circumference of the spheres via the total internal reflection at the polymer/air interface, and self-interferes to show sharp and periodic resonant PL lines. These resonating modes are so-called "whispering gallery modes" (WGMs). This work demonstrates how to measure WGM PL from single isolated spheres using the micro-photoluminescence (&#181;-PL) technique. In this technique, a focused laser beam irradiates a single microsphere, and the luminescence is detected by a spectrometer. A micromanipulation technique is then used to connect the microspheres one by one and to demonstrate the intersphere PL propagation and color conversion from coupled microspheres upon excitation at the perimeter of one sphere and detection of PL from the other microsphere. These techniques, &#181;-PL and micromanipulation, are useful for experiments on micro-optic application using polymer materials.

Protocols for the synthesis of microspheres from polymers, the manipulation of microspheres, and micro-photoluminescence measurements are presented.

This paper describes three methods of preparing fluorescent microspheres comprising &#960;-conjugated or non-conjugated polymers: vapor diffusion, ...

Engineering

Conventional BODIPY Conjugates for Live-Cell Super-Resolution Microscopy and Single-Molecule Tracking

Single molecule localization microscopy (SMLM) techniques overcome the optical diffraction limit of conventional fluorescence microscopy and can resolve intracellular structures and the dynamics of biomolecules with ~20 nm precision. A prerequisite for SMLM are fluorophores that transition from a dark to a fluorescent state in order to avoid spatio-temporal overlap of their point spread functions in each of the thousands of data acquisition frames. BODIPYs are well-established dyes with numerous conjugates used in conventional microscopy. The transient formation of red-shifted BODIPY ground-state dimers (DII) results in bright single molecule emission enabling single molecule localization microscopy (SMLM). Here we present a simple but versatile protocol for SMLM with conventional BODIPY conjugates in living yeast and mammalian cells. This procedure can be used to acquire super-resolution images and to track single BODIPY-DII states to extract spatio-temporal information of BODIPY conjugates. We apply this procedure to resolve lipid droplets (LDs), fatty acids, and lysosomes in living yeast and mammalian cells at the nanoscopic length scale. Furthermore, we demonstrate the multi-color imaging capability with BODIPY dyes when used in conjunction with other fluorescent&#160;probes. Our representative results show the differential spatial distribution and mobility of BODIPY-fatty acids and neutral lipids in yeast under fed and fasted conditions. This optimized protocol for SMLM can be used with hundreds of commercially available BODIPY conjugates and is a useful resource to study biological processes at the nanoscale far beyond the applications of this work.&#160;

Conventional BODIPY conjugates can be used for live-cell single-molecule localization microscopy (SMLM) through exploitation of their transiently forming, red-shifted ground state dimers. We present an optimized SMLM protocol to track and resolve subcellular neutral lipids and fatty acids in living mammalian and yeast cells at the nanoscopic length scale.

Single molecule localization microscopy (SMLM) techniques overcome the optical diffraction limit of conventional fluorescence microscopy and can resolve ...

Biochemistry

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 ...

Optical Control of a Neuronal Protein Using a Genetically Encoded Unnatural Amino Acid in Neurons

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to photo-regulate endogenous proteins expressed in their native environment. Here, we present an approach to optically control the function of a neuronal protein directly in neurons using a genetically encoded unnatural amino acid (Uaa). By using an orthogonal tRNA/aminoacyl-tRNA synthetase pair to suppress the amber codon, a photo-reactive Uaa 4,5-dimethoxy-2-nitrobenzyl-cysteine (Cmn) is site-specifically incorporated in the pore of a neuronal protein Kir2.1, an inwardly rectifying potassium channel. The bulky Cmn physically blocks the channel pore, rendering Kir2.1 non-conducting. Light illumination instantaneously converts Cmn into a smaller natural amino acid Cys, activating Kir2.1 channel function. We express these photo-inducible inwardly rectifying potassium (PIRK) channels in rat hippocampal primary neurons, and demonstrate that light-activation of PIRK ceases the neuronal firing due to the outflux of K+ current through the activated Kir2.1 channels. Using in utero electroporation, we also express PIRK in the embryonic mouse neocortex in vivo, showing the light-activation of PIRK in neocortical neurons. Genetically encoding Uaa imposes no restrictions on target protein type or cellular location, and a family of photoreactive Uaas is available for modulating different natural amino acid residues. This technique thus has the potential to be generally applied to many neuronal proteins to achieve optical regulation of different processes in brains. The current protocol presents an accessible procedure for intricate Uaa incorporation in neurons in vitro and in vivo to achieve photo control of neuronal protein activity on the molecular level.

Here, a procedure to selectively activate a neuronal protein with a short pulse of light by genetically encoding a photo-reactive unnatural amino acid into a target neuronal protein expressed in neurons in culture or in vivo is presented.

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to ...

Neuroscience

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 ...

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, ...

Control of Cell Geometry through Infrared Laser Assisted Micropatterning

Micropatterning is an established technique in the cell biology community used to study connections between the morphology and function of cellular compartments while circumventing complications arising from natural cell-to-cell variations. To standardize cell shape, cells are either confined in 3D molds or controlled for adhesive geometry through adhesive islands. However, traditional micropatterning techniques based on photolithography and deep UV etching heavily depend on clean rooms or specialized equipment. Here we present an infrared laser assisted micropatterning technique (microphotopatterning) modified from Doyle et al. that can be conveniently set up with commercially available imaging systems. In this protocol, we use a Nikon A1R MP+ imaging system to generate micropatterns with micron precision through an infrared (IR) laser that ablates preset regions on poly-vinyl alcohol coated coverslips. We employ a custom script to enable automated pattern fabrication with high efficiency and accuracy in systems not equipped with a hardware autofocus. We show that this IR laser assisted micropatterning (microphotopatterning) protocol results in defined patterns to which cells attach exclusively and take on the desired shape. Furthermore, data from a large number of cells can be averaged due to the standardization of cell shape. Patterns generated with this protocol, combined with high resolution imaging and quantitative analysis, can be used for relatively high throughput screens to identify molecular players mediating the link between form and function.

The protocol presented here enables automated fabrication of micropatterns that standardizes cell shape to study cytoskeletal structures within mammalian cells. This user-friendly technique can be set up with commercially available imaging systems and does not require specialized equipment inaccessible to standard cell biology laboratories.

Micropatterning is an established technique in the cell biology community used to study connections between the morphology and function of cellular ...

A Cardiac Microphysiological System for Studying Ca2+ Propagation via Non-genetic Optical Stimulation

In vitro cardiac microphysiological models are highly reliable for scientific research, drug development, and medical applications. Although widely accepted by the scientific community, these systems are still limited in longevity due to the absence of non-invasive stimulation techniques. Phototransducers provide an efficient stimulation method, offering a wireless approach with high temporal and spatial resolution while minimizing invasiveness in stimulation processes. In this manuscript, we present a fully optical method for stimulating and detecting the activity of an in vitro cardiac microphysiological model. Specifically, we fabricated engineered laminar anisotropic tissues by seeding human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) generated in a 3D bioreactor suspension culture. We employed a phototransducer, an amphiphilic azobenzene derivative, named Ziapin2, for stimulation and a Ca2+ dye (X-Rhod 1) for monitoring the system&#39;s response. The results demonstrate that Ziapin2 can photomodulate Ca2+ responses in the employed system without compromising tissue integrity, viability, or behavior. Furthermore, we showed that the light-based stimulation approach offers a similar resolution compared to electrical stimulation, the current gold standard. Overall, this protocol opens promising perspectives for the application of Ziapin2 and material-based photostimulation in cardiac research.

Using light to control cardiac cells and tissue enables non-contact stimulation, thereby preserving the natural state and function of the cells, making it a valuable approach for both basic research and therapeutic applications.

In vitro cardiac microphysiological models are highly reliable for scientific research, drug development, and medical applications. Although widely ...