This work was supported by a research grant from the German Federal Ministry of Education and Research (BMBF) (grant #13GW0043C) and and a European Office of Aerospace Research and Development (EOARD, grant # FA9550-15-1-0443)

1. RPE Cell Culture
<ol>
	<li>Isolation of RPE cells from porcine eyes
	<ol>
		<li>Obtain freshly enucleated porcine eyes from the local slaughterhouse. Keep them cool (4 &#176;C) and in a dark environment.</li>
		<li>Remove extracellular tissues with scissors and soak the eyes in an antiseptic solution for 5 min.</li>
		<li>Place the eyes in sterilized phosphate-buffered saline without calcium and magnesium (PBS (-)) until use.</li>
		<li>Using a scalpel, penetrate the sclera at about 5 mm posterior to the corneal limbus. Resect the whole anterior part of the eye by cutting with scissors all way through, parallel to the corneal limbus.</li>
		<li>Remove the anterior part of the eye (i.e., the cornea and lens) and the vitreous. Add 1 mL of PBS (-) and gently remove the neural retina. 
		NOTE: This &#34;eye cup,&#34; consisting of the sclera, choroid, and RPE, is now ready.</li>
		<li>Add pre-warmed (37 &#176;C) 0.25% trypsin in PBS (-) to the eye cup. Adjust the volume such that about 80% of the eye cup is filled with this trypsin solution.</li>
		<li>Incubate the eye cup with the trypsin solution in a 5% CO2 incubator at 37 &#176;C for 10 min.</li>
		<li>Remove the eye cup from the incubator and replace the 0.25% trypsin solution with a PBS (-) solution with 0.05% trypsin + 0.2% ethylenediaminetetraacetic acid tetrasodium salt (EDTA&#183;4N). Incubate the eye cup in the incubator for 45 min. 
		NOTE: After 45 min, the RPE cells will be either just loosely attached to the Bruch&#39;s membrane or already detached and floating in the trypsin-EDTA solution.</li>
		<li>Collect the RPE cells by gentle pipetting. Collect the cells and solution in a conical tube filled with 10 mL of culture medium (DMEM high glucose with L-glutamine), including 10% porcine serum, antibiotic/antimycotic, and sodium pyruvate (1 mM). 
		NOTE: The serum may neutralize the effect of trypsin.</li>
		<li>Centrifuge the cell suspension at 400 x g for 5 min at room temperature.</li>
		<li>Remove the supernatant and add 10 mL of fresh medium. Centrifuge again under the same conditions for 5 min.</li>
		<li>Remove the supernatant and add new medium, such that the cell concentration results in 5 x 105cells/mL (determined by counting the cells using a hemocytometer). Mix well by gentle pipetting.</li>
		<li>Distribute the cell suspension in cell culture dishes. Use 3 mL per 60 mm-diameter culture dish. 
		NOTE: This culture is called passage zero (P0).</li>
		<li>Maintain the cells in a 5% CO2 incubator at 37 &#176;C. Change half of the conditioned medium to fresh medium every second day.</li>
		<li>Subculture (step 1.2) if it becomes confluent.</li>
	</ol>
	</li>
	<li>Subculture of the RPE cell culture
	<ol>
		<li>Remove the culture medium and rinse the cells twice with PBS (-).</li>
		<li>Incubate the cells with PBS (-) solution with 0.05% trypsin + 0.2% EDTA in a 5% CO2 incubator at 37 &#176;C for 5 min.</li>
		<li>Detach the RPE cells by gentle pipetting and collect the cell suspension in a conical tube filled with 10 mL of culture medium, including 10% porcine serum.</li>
		<li>Centrifuge the cell suspension at 400 x g for 5 min at room temperature.</li>
		<li>Remove the supernatant and add new culture medium, making the cell concentration 5 x 105 cells/mL (determined by counting the cell number with a hemocytometer). Distribute the cells in new 60 mm-diameter culture dishes, as described in step 1.1.13. 
		NOTE: The cell culture is now passage 1 (P1).</li>
		<li>After confluency is reached, subculture the P1 culture to P2, using the same procedure described in steps 1.2.1-1.2.5. From the P2 culture, seed the cells on smaller culture dishes (30-mm inner diameter) instead of 60 mm-diameter culture dishes.</li>
		<li>For the experiments, use P2 or P3 cultures.</li>
	</ol>
	</li>
</ol>
2. Thulium Laser Irradiation
<ol>
	<li>Construction of the irradiation station
	<ol>
		<li>Connect a thulium laser device (1.94 &#181;m, power range: 0-20 W) to a 0.22-NA, 365-&#181;m core diameter fiber.</li>
		<li>Mechanically fix the fiber tip to the metal arm that is horizontally fixed to the vertical metal post of the irradiation station. Place the vertical post such that the tip of the laser fiber is located above the hot plate on which the cell culture dish is to be placed during irradiation.</li>
		<li>Lay a white paper on the hot plate and turn the aiming beam on (&#955; = 635 nm, max = 1 mW, diameter at paper level &#8776; 30 mm). Mark the circumference of the aiming beam on the white paper so that the position where the culture dish is to be placed during irradiation is known. 
		NOTE: The z-plane of the fiber tip may be changeable. Without any additional imaging optics, the laser spot diameter on the cell culture plane, placed 12 cm below the fiber tip, is about 30 mm, which is almost equivalent to the inner diameter of the cell culture dish. A schematic drawing of the setup is shown in Figure 1.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/54326/54326fig1.jpg" /> 
Figure 1: Schematic Image of the Thulium Laser Irradiation Station. A culture dish is placed on the heating plate. The cells are placed 12 cm below the thulium laser fiber tip so that the beam size is almost identical to the inner diameter of the culture dish (about 30 mm). The laser irradiation procedure is controlled by a time-controlled routine of the custom-made system design platform. The power setting must be determined before the irradiation program is started. <a href="http://cloudfront.jove.com/files/ftp_upload/54326/54326fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="2">
	<li>Laser irradiation of the cell culture
	<ol>
		<li>1 h before irradiation, replace the culture medium completely with 1.2 mL of fresh medium. 
		NOTE: This is a CRITICAL STEP and must be strictly followed.</li>
		<li>Place the irradiation station (i.e., the hot plate and the post with which to fix the laser fiber) on a clean bench.</li>
		<li>Remove the cell culture dish from the incubator and place it on the marked position on the hot plate (step 2.1.3).</li>
		<li>Wear protective glasses. Turn on the thulium laser. Set the power as desired on the laser device (tunable from 0 - 20 W). Turn the emission on.</li>
		<li>Start a system design platform that controls the laser irradiation and timing protocol (supplemental file).</li>
		<li>Immediately after placing the culture dish on the hot plate, click the &#34;pre-heating time&#34; to start the timer for 140 s (&#34;pre-heating time 1&#34;); this will keep the culture medium temperature at 37 &#176;C before irradiation. 
		NOTE: After 140 s, a beep sound will turn on, and the next timer (&#34;pre-heating time 2&#34;) will start automatically counting 8 s. During this 8 s, the examiner may open the culture dish. After 148 s of pre-heating, a 10 s-long laser irradiation on the cell culture will be conducted automatically. In case of emergency, equip the laser device with a force-quit button to stop the laser immediately. This is a CRITICAL STEP and must be strictly followed. A special point of caution relates to opening the cover of the dish just before irradiation, at the beginning of the 8 s pre-heating time. Opening the cover may cool down the medium surface very quickly.</li>
		<li>After irradiation, immediately place the cover back onto the culture dish, leave the culture dish on the hot plate for an additional 7 s, and place it back in the 5% CO2 incubator at 37 &#176;C.</li>
	</ol>
	</li>
	<li>Measurement of the temperature distribution at the cellular level (temperature calibration)
	<ol>
		<li>Make small holes (about 300 &#181;m in diameter) close to the bottom on four sides (every 90&#176;) of a 30 mm diameter culture dish (without cells); use the tip of a needle (20G) heated with a Bunsen burner. Seal the holes with electrical isolation tape from outside and make a small hole with a fine needle so that only a fine thermocouple (200 &#181;m in diameter) may be inserted through this hole under watertight conditions.</li>
		<li>At the outside of the culture dish bottom, draw 2 perpendicular diameters and set the crossing point (i.e., the center of the bottom side) as the coordinate zero (0). Mark every 3 mm radially to the outside out the dish (i.e., 0, 3, 6, 9, 12, and 15 mm) in each direction along the lines (Figure 2, blue dots); the number of points should be 21 in total.</li>
		<li>Fill the cell culture dish with 1.2 mL of new culture medium. Place the culture dish onto a hot plate at 37 &#176;C, insert a fine thermocouple (200 &#181;m in diameter) into the side hole, and place its sensitive tip onto a marked position to be measured.</li>
		<li>Wear protective glasses. Turn on the thulium laser and manually set the power (between 0 and 20 W, in 0.1-W increments) of the laser devise. 
		NOTE: For temperature calibration, measurements with the power in increments of 3 W should be sufficient.</li>
		<li>Turn on the system design platform and click the &#34;Start Temp. Acquisition&#34; button (supplemental file) to start the temperature measurement.</li>
		<li>Conduct same procedure as in step 2.2.6. 
		NOTE: The control program measures the temperature of the inserted thermocouple every 100 ms and shows the temperature progression during irradiation in the GUI.</li>
		<li>Conduct these procedures for all 21 measurement points and at different power settings. Repeat the whole procedure three times for all points and for all power settings to achieve reliable data.</li>
		<li>Export temperature data as csv data, which can eventually be converted to a spreadsheet. Average the maximal temperature at the end of irradiation for the triplicate measurements at every point. Average the values from the points on the same circle (4 points in total, except the central point).</li>
		<li>Plot the obtained averaged maximal temperature on a graph, making the distance from the center of the dish (mm) as the x-axis and the temperature increase (&#916;T, &#176;C) as the y-axis. Use the fit function of a mathematical software program to fit a Gaussian model to the raw data. Create a Gaussian fit temperature distribution.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/54326/54326fig2.jpg" /> 
Figure 2: The Points for Temperature Calibration in One Cell Culture Dish. The temperature data was measured in the center and at 5 radial points over 4 different angles (blue dots). <a href="http://cloudfront.jove.com/files/ftp_upload/54326/54326fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Biological Assessments for Cell Responses after Different Thermal Irradiations
<ol>
	<li>Assessment of cell viability ( i.e., living, apoptotic, and dead) following different power settings and the determination of the cell death threshold
	<ol>
		<li>At the indicated time points (i.e., 3, 24, and 48 h after irradiation), wash the cells with PBS (-) and use a commercially available kit to assess cell viability (i.e., vital, apoptotic, dead) according to the manufacturer&#39;s protocol.</li>
		<li>Prepare a staining solution by adding 5 &#181;L of fluorescein isothiocyanate (FITC)-annexin V, 5 &#181;L of ethidium homodimer III, and 5 &#181;L of Hoechst 33342 to 100 &#181;L of 1x binding buffer (all are kit components). Prepare enough staining solution to cover the cells. Incubate the cells for 15 min.</li>
		<li>Wash the cell culture with the binding buffer twice, replace the binding buffer with PBS (-), and set the culture on the stage of a fluorescence microscope.</li>
		<li>Switch the light path to the ocular lens, select the 4&#39;,6-Diamidin-2-phenylindol (DAPI) filter, turn on the illumination light, and find the focused plane with the 4x objective.</li>
		<li>Change the light path to the camera, find the image on the computer screen in the microscope imaging software, and adjust the focus.</li>
		<li>Use the stitch function (i.e., the function to record multiple images across the dish and then create a single, large image) of the microscope-specific software to obtain the fluorescence image of the whole cell culture dish. Use 3 different filter sets-DAPI, FITC, and tetramethylrhodamine (TRITC)-to image Hoechst 33342-positive cells (all cell nuclei), FITC-annexin V-positive cells (apoptotic), and ethidium homodimer III-positive cells (dead), respectively.</li>
		<li>Measure the radius (mm) of the dead (ethidium homodimer III-positive) region and the outer/inner radius of the apoptotic (annexin V-positive) band-form region in the stained cell cultures. Apply these radii to the fitted Gaussian function of the temperature distribution for the corresponding power setting. Calculate the exact temperature at the rim of the dead or apoptotic region to clarify the threshold temperatures for cell death and apoptotic change.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Inagaki, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+efficacy+of+pure+yellow+(577-nm)+and+810-nm+subthreshold+micropulse+laser+photocoagulation+combined+with+yellow+(561-577-nm)+direct+photocoagulation+for+diabetic+macular+edema.">Comparative efficacy of pure yellow (577-nm) and 810-nm subthreshold micropulse laser photocoagulation combined with yellow (561-577-nm) direct photocoagulation for diabetic macular edema.</a> Jpn J Ophthalmol. 59 (1), 21-28 (2015).</li><li>Roider, 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=Selective+retina+therapy+(SRT)+for+clinically+significant+diabetic+macular+edema.">Selective retina therapy (SRT) for clinically significant diabetic macular edema.</a> Graefes Arch Clin Exp Ophthalmol. 248 (9), 1263-1272 (2010).</li><li>Brinkmann, 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=Real-time+temperature+determination+during+retinal+photocoagulation+on+patients.">Real-time temperature determination during retinal photocoagulation on patients.</a> J Biomed Opt. 17 (6), 061219 (2012).</li><li>Yoshimura, 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=Photocoagulated+human+retinal+pigment+epithelial+cells+produce+an+inhibitor+of+vascular+endothelial+cell+proliferation.">Photocoagulated human retinal pigment epithelial cells produce an inhibitor of vascular endothelial cell proliferation.</a> Invest Ophthalmol Vis Sci. 36 (8), 1686-1691 (1995).</li><li>Denton, M. L., 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=Damage+Thresholds+for+Exposure+to+NIR+and+Blue+Lasers+in+an+In+Vitro+RPE+Cell+System.">Damage Thresholds for Exposure to NIR and Blue Lasers in an In Vitro RPE Cell System.</a> Invest Ophthalmol Vis Sci. 47 (7), 3065-3073 (2006).</li><li>Shrestha, R., Choi, T. Y., Chang, W., Kim, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-precision+micropipette+sensor+for+cellular-level+real-time+thermal+characterization.">A high-precision micropipette sensor for cellular-level real-time thermal characterization.</a> Sensors (Basel). 11 (9), 8826-8835 (2011).</li><li>Gao, F., Ye, Y., Zhang, Y., Yang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Water+bath+hyperthermia+reduces+stemness+of+colon+cancer+cells.">Water bath hyperthermia reduces stemness of colon cancer cells.</a> Clin Biochem. 46 (16-17), 1747-1750 (2013).</li><li>Jansen, E. D., van Leeuwen, T. G., Motamedi, M., Borst, C., Welch, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature+dependence+of+the+absorption+coefficient+of+water+for+midinfrared+laser+radiation.">Temperature dependence of the absorption coefficient of water for midinfrared laser radiation.</a> Lasers Surg Med. 14 (3), 258-268 (1994).</li><li>Iwami, H., Pruessner, J., Shiraki, K., Brinkmann, R., Miura, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+effect+of+a+laser-induced+sub-lethal+temperature+rise+on+RPE+cells+from+oxidative+stress.">Protective effect of a laser-induced sub-lethal temperature rise on RPE cells from oxidative stress.</a> Exp Eye Res. 124, 37-47 (2014).</li><li>Denton, M. L., 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=Spatially+correlated+microthermography+maps+threshold+temperature+in+laser-induced+damage.">Spatially correlated microthermography maps threshold temperature in laser-induced damage.</a> J Biomed Optics. 16 (3), (2011).</li><li>Morgan, C. M., Schatz, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atrophic+creep+of+the+retinal+pigment+epithelium+after+focal+macular+photocoagulation.">Atrophic creep of the retinal pigment epithelium after focal macular photocoagulation.</a> Ophthalmology. 96 (1), 96-103 (1989).</li></ol>

The authors have nothing to disclose.

In discussing temperature-related biological cellular responses, not only the temperature, but also the time duration of the increased temperature, is of importance, since most biochemical processes are time-dependent. Particularly in the field of laser-induced hyperthermia in ophthalmology, due to the short time range-from milliseconds to seconds-it is difficult to investigate cellular thermal effects with precise temperature control. Therefore, a laser irradiation setup suitable for the cell culture model and with an o...

Understanding temperature-dependent cell biological responses is of great importance to successful hyperthermia treatments. Retinal laser photocoagulation with a thermal laser, used in ophthalmology, is one of the most established laser treatments in medicine. Visible light, mostly from green to yellow wavelengths, is used in retinal laser treatment. The light is highly absorbed by the melanin in retinal pigment epithelial (RPE) cells, which form the outermost cell monolayer of the retina. There has been recent interest among physicians and researchers in very mild thermal irradiation (sub-visible photocoagulation) as a new therapeutic strategy for different kinds of retinal disorders1,2. Following this trend, our interest is in sub-lethally heating RPE cells under precise temperature control, a technique called temperature-controlled photothermal therapy (TC-PTT).
Recent optoacoustic technology from our institute has allowed for the real-time measurement of temperature increases at irradiated sites in the retina. This enables control over the temperature increase during irradiation3. However, since sub-lethal hyperthermia on the retina, caused by heating RPE cells sub-lethally, has not been previously considered due to the impossibility of measuring and controlling the temperature, the temperature-dependent cell responses of RPE cells following thermal laser irradiation has been studied very little to date. Moreover, not only has the temperature difference not been discussed in detail, but also the difference in the cell behavior of the surviving cells after sub-lethal and lethal irradiation. Therefore, to gather scientific evidence on TC-PTT-based treatments, we aim to elucidate the temperature-dependent RPE cell biological responses and their mechanisms using in vitro experimental setups.
For this purpose, it is necessary to establish a cell-heating setup that meets the following conditions: 1) a possibility for fast temperature increases, 2) a precisely controlled time and temperature, and 3) a relatively high number of examined cells for biological experiments. Regarding the heating method, a clinical laser, such as a frequency-doubled Nd.YAG laser (532 nm), is unfortunately unsuitable for cell culture heating. This is because of the strongly reduced number of melanosomes in cultured RPE cells. The laser light absorption might be inhomogeneous, and the temperature increase at the cellular level is variable between experiments, even when irradiated with same radiation power. Several previous studies have reported the use of black paper beneath the dish bottom during irradiation4 or the use of additional melanosomes that are phagocytized by the culture cells before the experiments5,6. Many of the in vitro biological studies to assess hyperthermia-induced cell responses have been performed using a hot plate, a water bath, or a CO2 incubator with a temperature setting7. These methods require a long heating period because it takes some time (i.e., several minutes) to reach the desired temperature. Furthermore, using these methods, it is difficult to obtain a detailed thermal history (i.e., temperature multiplied by time) at the cellular level. Moreover, the temperature among the cells at different positions in one culture dish may differ due to variable temperature diffusion. In most cases, this temporal and spatial temperature information during hyperthermia has not been taken into consideration for biological analyses, even though biological cell response may be critically affected by the temperature and the time duration of the increased temperature.
To overcome these problems, a continuous-wave thulium laser was used here to heat the cells. Thulium laser radiation (&#955; = 1.94 &#181;m) is strongly absorbed by water8, and the cells at the bottom of the culture dish are thermally stimulated solely through thermal diffusion. The laser fiber with a 365-&#181;m diameter is set about 12 cm above the culture dish, without any optics in between. The laser beam diameter diverges such that it is almost equivalent to the inner diameter of the culture dish (30 mm) at the surface of the culture medium.With a consistent amount of culture medium, it is possible to irradiate the cells with the temperature increase of high repeatability. Variable power settings enable irradiation with up to 20 W, and the medium temperature at the cellular level may be increased up to &#916;T &#8776; 26 &#176;C in 10 s.
By modifying the irradiation conditions, it is also possible to change the laser beam profile to vary the temperature distribution in a culture dish. For example, it is possible to investigate with a Gaussian-like temperature distribution, as in the current study, or with a homogeneous temperature distribution. The latter may be advantageous for investigating the effects of temperature-dependent cell responses more specifically for sub-lethal temperature increases, but not for cell death stress or wound healing responses.
Altogether, thulium laser irradiation may enable the investigation of different kinds of biological factors, such as gene/protein expression, cell death kinetics, cell proliferation, and cell functionality development, after different thermal exposures.

<table><tbody><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle&amp;rsquo;s Medium - high glucose</td><td>Sigma-Aldrich</td><td>D5796-500ML</td><td>Add (2)-(4) before use. Warm in 37 &amp;deg;C water bath before use.</td></tr><tr><td>Antibiotic Antimycotic Solution (100 &amp;times;)</td><td>Sigma-Aldrich</td><td>A5955-100ML</td><td>Containing 10,000 units penicillin, 10 mg streptomycin and 25 &amp;mu;g Amphotericin B in 1mL. Add 5.5 mL in 500 mL medium bottle (1) before use.</td></tr><tr><td>Sodium pyruvate (100 mM)</td><td>Sigma-Aldrich</td><td>S8636-100ML</td><td>Add 5.5 mL in 500 mL medium bottle (1) before use (final concentration: 1 mM)</td></tr><tr><td>Porcine serum</td><td>Sigma-Aldrich</td><td>12736C-500ML</td><td>Add 50 mL in 500 mL medium bottole (1) before use (final: 10%)</td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>Sigma-Aldrich</td><td>D8537-500ML</td><td></td></tr><tr><td>Trypsin from porcine pancreas</td><td>Sigma-Aldrich</td><td>T4799-25G</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA)</td><td>Sigma-Aldrich</td><td>ED-100G</td><td></td></tr><tr><td>Human VEGF Quantikine ELISA Kit</td><td>R&amp;amp;D System</td><td>DVE00</td><td></td></tr><tr><td>Oxiselect Total Glutathione Assay Kit</td><td>Cell Biolabs, Inc</td><td>STA-312</td><td></td></tr><tr><td>Apoptotic/Necrotic/Healthy Cells Detection Kit</td><td>PromoKine</td><td>PK-CA707-30018</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipments&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Thulium laser</td><td>Starmedtec GmbH</td><td>Prototype</td><td>0-20 W</td></tr><tr><td>365 mm core diameter fiber</td><td>LASER COMPONENTS Germany</td><td>CF01493-52</td><td></td></tr><tr><td>Thermocouple</td><td>Omega Engineering Inc</td><td>HYP-0- 33-1-T-G-60-SMPW-M</td><td></td></tr><tr><td>Heating plate</td><td>MEDAX</td><td></td><td></td></tr><tr><td>Microplate reader (spectrofluorometer)</td><td>Molecular Device</td><td>Spectramax M4</td><td></td></tr><tr><td>cell homogenizer</td><td>QIAGEN</td><td>TissueLyser LT</td><td></td></tr><tr><td>Fluorescence microscope</td><td>Nikon</td><td>ECLIPSE Ti</td><td></td></tr><tr><td>mathematical software program</td><td>The Mathworks. Inc</td><td>MATLAB Release 2015b</td><td></td></tr><tr><td>system-design platform</td><td>National Instrument</td><td>Labview</td><td>Laboratory Virtual Instrument Engineering Workbench</td></tr></tbody></table>

continuous-wave thulium laser for heating cultured cells to investigate cellular thermal effects

An original method to heat cultured cells using a 1.94 &#181;m continuous-wave thulium laser for biological assessment is introduced here. Thulium laser radiation is strongly absorbed by water, and the cells at the bottom of the culture dish are heated through thermal diffusion. A laser fiber with a diameter of 365 &#181;m is set about 12 cm above the culture dish, without any optics, such that the laser beam diameter is almost equivalent to the inner diameter of the culture dish (30 mm). By keeping a consistent amount of culture medium in each experiment, it is possible to irradiate the cells with a highly reproducible temperature increase.
To calibrate the temperature increase and its distribution in one cell culture dish for each power setting, the temperature was measured during 10 s of irradiation at different positions and at the cellular level. The temperature distribution was represented using a mathematical graphics software program, and its pattern across the culture dish was in Gaussian form. After laser irradiation, different biological experiments could be performed to assess temperature-dependent cell responses. In this manuscript, viability staining (i.e., distinguishing live, apoptotic, and dead cells) is introduced to help determine the threshold temperatures for cell apoptosis and death after different points in time.
The advantages of this method are the preciseness of the temperature and the time of heating, as well as its high efficiency in heating cells in a whole cell culture dish. Furthermore, it allows for study with a wide variety of temperatures and time durations, which can be well-controlled by a computerized operating system.

Temperature distribution after different power settings
All temperature developments for each single irradiation were monitored in the temperature calibration. From this data, the maximal temperature at the measured point was obtained and defined as Tmax (&#176;C). As shown in Figure 3A, the program was executed at the time point when the culture dish was placed on the heating plate. After t...

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Continuous-wave Thulium Laser for Heating Cultured Cells to Investigate Cellular Thermal Effects

An original experimental setup for heating cells in a culture dish using 1.94 &#181;m continuous-wave laser radiation is introduced here. Using this method, the biological responses of retinal pigment epithelial (RPE) cells after different thermal exposures can be investigated.

An original method to heat cultured cells using a 1.94 &#181;m continuous-wave thulium laser for biological assessment is introduced here. Thulium laser ...

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7.7K Views.  University of Luebeck. An original experimental setup for heating cells in a culture dish using 1.94 µm continuous-wave laser radiation is introduced here. Using this method, the biological responses of retinal pigment epithelial (RPE) cells after different thermal exposures can be investigated.

Video: Continuous-wave Thulium Laser for Heating Cultured Cells to Investigate Cellular Thermal Effects

Application of Laser Micro-irradiation for Examination of Single and Double Strand Break Repair in Mammalian Cells

Highly coordinated DNA repair pathways exist to detect, excise and replace damaged DNA bases, and coordinate repair of DNA strand breaks. While molecular biology techniques have clarified structure, enzymatic functions, and kinetics of repair proteins, there is still a need to understand how repair is coordinated within the nucleus. Laser micro-irradiation offers a powerful tool for inducing DNA damage and monitoring the recruitment of repair proteins. Induction of DNA damage by laser micro-irradiation can occur with a range of wavelengths, and users can reliably induce single strand breaks, base lesions and double strand breaks with a range of doses. Here, laser micro-irradiation is used to examine repair of single and double strand breaks induced by two common confocal laser wavelengths, 355 nm and 405 nm. Further, proper characterization of the applied laser dose for inducing specific damage mixtures is described, so users can reproducibly perform laser micro-irradiation data acquisition and analysis.

Confocal fluorescence microscopy and laser micro-irradiation offer tools for inducing DNA damage and monitoring the response of DNA repair proteins in selected sub-nuclear areas. This technique has significantly advanced our knowledge of damage detection, signaling, and recruitment. This manuscript demonstrates these technologies to examine single and double strand break repair.

Highly coordinated DNA repair pathways exist to detect, excise and replace damaged DNA bases, and coordinate repair of DNA strand breaks. While molecular ...

Cancer Research

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation became a valuable experimental tool to investigate the DNA damage response (DDR) in vivo. It allows real-time high-resolution single-cell analysis of macromolecular dynamics in response to laser-induced damage confined to a submicrometer region in the cell nucleus. However, various laser conditions have been used without appreciation of differences in the types of damage induced. As a result, the nature of the damage is often not well characterized or controlled, causing apparent inconsistencies in the recruitment or modification profiles. We demonstrated that different irradiation conditions (i.e., different wavelengths as well as different input powers (irradiances) of a femtosecond (fs) near-infrared (NIR) laser) induced distinct DDR and repair protein assemblies. This reflects the type of DNA damage produced. This protocol describes how titration of laser input power allows induction of different amounts and complexities of DNA damage, which can easily be monitored by detection of base and crosslinking damages, differential poly (ADP-ribose) (PAR) signaling, and pathway-specific repair factor assemblies at damage sites. Once the damage conditions are determined, it is possible to investigate the effects of different damage complexity and differential damage signaling as well as depletion of upstream factor(s) on any factor of interest.

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

The goal of this protocol is to describe how to use laser microirradiation to induce different types of DNA damage, including relatively simple strand breaks and complex damage, to study DNA damage signaling and repair factor assembly at damage sites in vivo.

DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation ...

Genetics

High-resolution Thermal Micro-imaging Using Europium Chelate Luminescent Coatings

Micro-electronic devices often undergo significant self-heating when biased to their typical operating conditions. This paper describes a convenient optical micro-imaging technique which can be used to map and quantify such behavior. Europium thenoyltrifluoroacetonate (EuTFC) has a 612 nm luminescence line whose activation efficiency drops strongly with increasing temperature, due to T-dependent interactions between the Eu3+ ion and the organic chelating compound. This material may be readily coated on to a sample surface by thermal sublimation in vacuum. When the coating is excited with ultraviolet light (337 nm) an optical micro-image of the 612 nm luminescent response can be converted directly into a map of the sample surface temperature. This technique offers spatial resolution limited only by the microscope optics (about 1 micron) and time resolution limited by the speed of the camera employed. It offers the additional advantages of only requiring comparatively simple and non-specialized equipment, and giving a quantitative probe of sample temperature.

Europium thenoyltrifluoroacetonate (EuTFC) has an optical luminescence line at 612 nm, whose activation efficiency decreases strongly with temperature. If a sample coated with a thin film of this material is micro-imaged, the 612 nm luminescent response intensity may be converted into a direct map of sample surface temperature.

Micro-electronic devices often undergo significant self-heating when biased to their typical operating conditions. This paper describes a convenient ...

Engineering

One Minute, Sub-One-Watt Photothermal Tumor Ablation Using Porphysomes, Intrinsic Multifunctional Nanovesicles

We recently developed porphysomes as intrinsically multifunctional nanovesicles. A photosensitizer, pyropheophorbide &alpha;, was conjugated to a phospholipid and then self-assembled to liposome-like spherical vesicles. Due to the extremely high density of porphyrin in the porphyrin-lipid bilayer, porphysomes generated large extinction coefficients, structure-dependent fluorescence self-quenching, and excellent photothermal efficacy. In our formulation, porphysomes were synthesized using high pressure extrusion, and displayed a mean particle size around 120 nm. Twenty-four hr post-intravenous injection of porphysomes, the local temperature of the tumor increased from 30 &deg;C to 62 &deg;C rapidly upon one minute exposure of 750 mW (1.18 W/cm2), 671 nm laser irradiation. Following the complete thermal ablation of the tumor, eschars formed and healed within 2 weeks, while in the control groups the tumors continued to grow and all reached the defined end point within 3 weeks. These data show how porphysomes can be used as potent photothermal therapy (PTT) agents.

We developed novel intrinsic multifunctional nanovesicles called porphysomes, which have structure-dependent fluorescence self-quenching and unique photothermal properties, thus functioning as potent photothermal therapy agents. We formulated porphysomes using high pressure extrusion and investigated their photothermal therapy efficacy in a xenograft tumor model.

We recently developed porphysomes as intrinsically multifunctional nanovesicles. A photosensitizer, pyropheophorbide &alpha;, was conjugated to a ...

Bioengineering

Subsurface Defect Localization by Structured Heating Using Laser Projected Photothermal Thermography

The presented method is used to locate subsurface defects oriented perpendicularly to the surface. To achieve this, we create destructively interfering thermal wave fields that are disturbed by the defect. This effect is measured and used to locate the defect. We form the destructively interfering wave fields by using a modified projector. The original light engine of the projector is replaced with a fiber-coupled high-power diode laser. Its beam is shaped and aligned to the projector&#39;s spatial light modulator and optimized for optimal optical throughput and homogeneous projection by first characterizing the beam profile, and, second, correcting it mechanically and numerically. A high-performance infrared (IR) camera is set up according to the tight geometrical situation (including corrections of the geometrical image distortions) and the requirement to detect weak temperature oscillations at the sample surface. Data acquisition can be performed once a synchronization between the individual thermal wave field sources, the scanning stage, and the IR camera is established by using a dedicated experimental setup which needs to be tuned to the specific material being investigated. During data post-processing, the relevant information on the presence of a defect below the surface of the sample is extracted. It is retrieved from the oscillating part of the acquired thermal radiation coming from the so-called depletion line of the sample surface. The exact location of the defect is deduced from the analysis of the spatial-temporal shape of these oscillations in a final step. The method is reference-free and very sensitive to changes within the thermal wave field. So far, the method has been tested with steel samples but is applicable to different materials as well, in particular to temperature sensitive materials.

This method aims at locating vertical subsurface defects. Here, we couple a laser with a spatial light modulator and trigger its video input to heat a sample surface deterministically with two anti-phased modulated lines while acquiring highly resolved thermal images. The defect position is retrieved from evaluating thermal wave interference minima.

The presented method is used to locate subsurface defects oriented perpendicularly to the surface. To achieve this, we create destructively interfering ...

Custom-designed Laser-based Heating Apparatus for Triggered Release of Cisplatin from Thermosensitive Liposomes with Magnetic Resonance Image Guidance

Liposomes have been employed as drug delivery systems to target solid tumors through exploitation of the enhanced permeability and retention (EPR) effect resulting in significant reductions in systemic toxicity. Nonetheless, insufficient release of encapsulated drug from liposomes has limited their clinical efficacy. Temperature-sensitive liposomes have been engineered to provide site-specific release of drug in order to overcome the problem of limited tumor drug bioavailability. Our lab has designed and developed a heat-activated thermosensitive liposome formulation of cisplatin (CDDP), known as HTLC, to provide triggered release of CDDP at solid tumors. Heat-activated delivery in vivo was achieved in murine models using a custom-built laser-based heating apparatus that provides a conformal heating pattern at the tumor site as confirmed by MR thermometry (MRT). A fiber optic temperature monitoring device was used to measure the temperature in real-time during the entire heating period with online adjustment of heat delivery by alternating the laser power. Drug delivery was optimized under magnetic resonance (MR) image guidance by co-encapsulation of an MR contrast agent (i.e.,&#160;gadoteridol) along with CDDP into the thermosensitive liposomes as a means to validate the heating protocol and to assess tumor accumulation. The heating protocol consisted of a preheating period of 5 min prior to administration of HTLC and 20 min heating post-injection. This heating protocol resulted in effective release of the encapsulated agents with the highest MR signal change observed in the heated tumor in comparison to the unheated tumor and muscle. This study demonstrated the successful application of the laser-based heating apparatus for preclinical thermosensitive liposome development and the importance of MR-guided validation of the heating protocol for optimization of drug delivery.

AN MRI-compatible&#160;custom-designed laser-based heating apparatus has been developed to provide local heating of subcutaneous tumors in order to activate release of agents from thermosensitive liposomes specifically at the tumor region.

Liposomes have been employed as drug delivery systems to target solid tumors through exploitation of the enhanced permeability and retention (EPR) effect ...

Whole Cell Patch Clamp for Investigating the Mechanisms of Infrared Neural Stimulation

It has been demonstrated in recent years that pulsed, infrared laser light can be used to elicit electrical responses in neural tissue, independent of any further modification of the target tissue. Infrared neural stimulation has been reported in a variety of peripheral and sensory neural tissue in vivo, with particular interest shown in stimulation of neurons in the auditory nerve. However, while INS has been shown to work in these settings, the mechanism (or mechanisms) by which infrared light causes neural excitation is currently not well understood. The protocol presented here describes a whole cell patch clamp method designed to facilitate the investigation of infrared neural stimulation in cultured primary auditory neurons. By thoroughly characterizing the response of these cells to infrared laser illumination in vitro under controlled conditions, it may be possible to gain an improved understanding of the fundamental physical and biochemical processes underlying infrared neural stimulation.

Infrared nerve stimulation has been proposed as an alternative to electrical stimulation in a range of nerve types, including those associated with the auditory system. This protocol describes a patch clamp method for studying the mechanism of infrared nerve stimulation in a culture of primary auditory neurons.

It has been  demonstrated in recent years that pulsed, infrared laser light can be used to  elicit electrical responses in neural tissue, independent of ...

Neuroscience

Magnetic-, Acoustic-, and Optical-Triple-Responsive Microbubbles for Magnetic Hyperthermia and Pothotothermal Combination Cancer Therapy

The precision delivery of anti-cancer agents which aim for targeted and deep-penetrated delivery as well as a controlled release at the tumor site has been challenged. Here, we fabricate iron oxide nanoparticle shelled microbubbles (NSMs) through self-assembly, synergizing magnetic, acoustic, and optical responsiveness in one nanotherapeutic platform. Iron oxide nanoparticles serve as both magnetic and photothermal agents. Once intravenously injected, NSMs can be magnetically guided to the tumor site. Ultrasound triggers the release of iron oxide nanoparticles, facilitating the penetration of nanoparticles deep into the tumor due to the cavitation effect of microbubbles. Thereafter, magnetic hyperthermia and photothermal therapy can be performed on the tumor for combinational cancer therapy, a solution for cancer resistance due to the tumor heterogeneity. In this protocol, the synthesis and characterization of NSMs including structural, chemical, magnetic and acoustic properties were performed. In addition, the anti-cancer efficacy by thermal therapy was investigated using in vitro cell cultures. The proposed delivery strategy and combination therapy holds great promise in cancer treatment to improve both delivery and anticancer efficacies.

Presented here is a protocol for the fabrication of iron oxide nanoparticle-shelled microbubbles (NSMs) through self-assembly, synergizing magnetic, acoustic, and optical responsiveness in one nanotherapeutic platform for magnetic hyperthermia and photothermal combination cancer therapy.

The precision delivery of anti-cancer agents which aim for targeted and deep-penetrated delivery as well as a controlled release at the tumor site has ...

Photostimulation by Femtosecond Laser Activates Extracellular-signal-regulated Kinase (ERK) Signaling or Mitochondrial Events in Target Cells

Direct control of cellular defined molecular events is important to life science. Recently, studies have demonstrated that femtosecond laser stimulation can simultaneously activate multiple cellular molecular signaling pathways. In this protocol, we show that through coupling femtosecond laser into a confocal microscope, cells can be stimulated precisely by the tightly-focused laser. Some molecular processes that can be simultaneously observed are subsequently activated. We present detailed protocols of the photostimulation to activate extracellular signal regulated kinase (ERK) signaling pathway in Hela cells. Mitochondrial flashes of reactive oxygen species (ROS) and other mitochondrial events can be also stimulated if focusing the femtosecond laser pulse on a certain mitochondrial tubular structure. This protocol includes pretreating cells before photostimulation, delivering the photostimulation by a femtosecond laser flash onto the target, and observing/identifying molecular changes afterwards. This protocol represents an all-optical tool for related biological researches.

Photostimulation by Femtosecond Laser Activates Extracellular-signal-regulated Kinase ERK Signaling or Mitochondrial Events in Target Cells

Tightly-focused femtosecond laser can deliver precise stimulation to cells by being coupled into a confocal microscopy enabling the real-time observation and photostimulation. The photostimulation can activate cell molecular events including ERK signaling pathway and mitochondrial flashes of reactive oxygen species.

Direct control of cellular defined molecular events is important to life science. Recently, studies have demonstrated that femtosecond laser stimulation ...

Photobiomodulation Growth Augmentation of Hydrogel Embedded Stem Cells 

Three&#45;Dimensional Cell Culture of Adipose-Derived Stem Cells in a Hydrogel with Photobiomodulation Augmentation

Adipose-derived stem cells (ADSCs), possessing multipotent mesenchymal characteristics akin to stem cells, are frequently employed in regenerative medicine due to their capacity for a diverse range of cell differentiation and their ability to enhance migration, proliferation, and mitigate inflammation. However, ADSCs often face challenges in survival and engraftment within wounds, primarily due to unfavorable inflammatory conditions. To address this issue, hydrogels have been developed to sustain ADSC viability in wounds and expedite the wound healing process. Here, we aimed to assess the synergistic impact of photobiomodulation (PBM) on ADSC proliferation and cytotoxicity within a 3D cell culture framework. Immortalized ADSCs were seeded into 10 &#181;L hydrogels at a density of 2.5 x 103 cells and subjected to irradiation using 525 nm and 825 nm diodes at fluencies of 5 J/cm2 and 10 J/cm2. Morphological changes, cytotoxicity, and proliferation were evaluated at 24 h and 10 days post-PBM exposure. The ADSCs exhibited a rounded morphology and were dispersed throughout the gel as individual cells or spheroid aggregates. Importantly, both PBM and 3D culture framework displayed no cytotoxic effects on the cells, while PBM significantly enhanced the proliferation rates of ADSCs. In conclusion, this study demonstrates the use of hydrogel as a suitable 3D environment for ADSC culture and introduces PBM as a significant augmentation strategy, particularly addressing the slow proliferation rates associated with 3D cell culture.

Author Spotlight: Advancements in Stem Cell Regenerative Therapy Through Photobiomodulation

Here, we present a protocol demonstrating the use of hydrogel as a three-dimensional (3D) cell culture framework for adipose-derived stem cell (ADSC) culture and introducing photobiomodulation (PBM) to enhance the proliferation of ADSCs within the 3D culture setting.

Continuous-wave Thulium Laser for Heating Cultured Cells to Investigate Cellular Thermal Effects

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Chapters in this video

Application of Laser Micro-irradiation for Examination of Single and Double Strand Break Repair in Mammalian Cells

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

High-resolution Thermal Micro-imaging Using Europium Chelate Luminescent Coatings

One Minute, Sub-One-Watt Photothermal Tumor Ablation Using Porphysomes, Intrinsic Multifunctional Nanovesicles

Subsurface Defect Localization by Structured Heating Using Laser Projected Photothermal Thermography

Custom-designed Laser-based Heating Apparatus for Triggered Release of Cisplatin from Thermosensitive Liposomes with Magnetic Resonance Image Guidance

Whole Cell Patch Clamp for Investigating the Mechanisms of Infrared Neural Stimulation

Magnetic-, Acoustic-, and Optical-Triple-Responsive Microbubbles for Magnetic Hyperthermia and Pothotothermal Combination Cancer Therapy

Photostimulation by Femtosecond Laser Activates Extracellular-signal-regulated Kinase (ERK) Signaling or Mitochondrial Events in Target Cells

Three-Dimensional Cell Culture of Adipose-Derived Stem Cells in a Hydrogel with Photobiomodulation Augmentation