We would like to thank Prof. Ferenc Krausz for the discussions and Najd Altwaijry for her support for finalizing the manuscript. This work has been financed by the Centre for Advanced Laser Applications (CALA).

<ol>
	<li>Fattahi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Third-generation+femtosecond+technology.">Third-generation femtosecond technology.</a> Optica. 1 (1), 45-63 (2014).</li><li>Hentschel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attosecond+metrology.">Attosecond metrology.</a> Nature. 414 (6863), 509-513 (2001).</li><li>Cavalieri, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intense+1.5-cycle+near+infrared+laser+waveforms+and+their+use+for+the+generation+of+ultra-broadband+soft-x-ray+harmonic+continua.">Intense 1.5-cycle near infrared laser waveforms and their use for the generation of ultra-broadband soft-x-ray harmonic continua.</a> New J Phys. 9 (7), 242 (2007).</li><li>Schweinberger, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Waveform-controlled+near-single-cycle+milli-joule+laser+pulses+generate+sub-10+nm+extreme+ultraviolet+continua.">Waveform-controlled near-single-cycle milli-joule laser pulses generate sub-10 nm extreme ultraviolet continua.</a> Opt Lett. 37, 3573-3575 (2012).</li><li>Buck, A. <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+observation+of+laser-driven+electron+acceleration.">Real-time observation of laser-driven electron acceleration.</a> Nature Phys. 7 (7), 543-548 (2011).</li><li>Zhong, H., Karpowicz, N., Zhang, X. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Terahertz+emission+profile+from+laser-induced+air+plasma.">Terahertz emission profile from laser-induced air plasma.</a> Appl Phys Lett. 88 (26), 261103 (2006).</li><li>Herrmann, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+sub-three-cycle,+16+TW+light+pulses+by+using+noncollinear+optical+parametric+chirped-pulse+amplification.">Generation of sub-three-cycle, 16 TW light pulses by using noncollinear optical parametric chirped-pulse amplification.</a> Opt Lett. 34 (16), 2459-2461 (2009).</li><li>Adachi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=1.5+mJ,+6.4+fs+parametric+chirped-pulse+amplification+system+at+1+kHz.">1.5 mJ, 6.4 fs parametric chirped-pulse amplification system at 1 kHz.</a> Opt Lett. 32 (17), 2487-2489 (2007).</li><li>Adachi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=5-fs,+multi-mJ,+CEP-locked+parametric+chirped-pulse+amplifier+pumped+by+a+450-nm+source+at+1+kHz.">5-fs, multi-mJ, CEP-locked parametric chirped-pulse amplifier pumped by a 450-nm source at 1 kHz.</a> Opt express. 16 (19), 14341-14352 (2008).</li><li>Yin, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-efficiency+optical+parametric+chirped-pulse+amplifier+in+BiB3O6+for+generation+of+3+mJ,+two-cycle,+carrier-envelope-phase-stable+pulses+at+1.7+&#181;m.">High-efficiency optical parametric chirped-pulse amplifier in BiB3O6 for generation of 3 mJ, two-cycle, carrier-envelope-phase-stable pulses at 1.7 &#181;m.</a> Opt Lett. 41 (6), 1142-1145 (2016).</li><li>Deng, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carrier-envelope-phase-stable,+1.2+mJ,+1.5+cycle+laser+pulses+at+2.1+&#181;m.">Carrier-envelope-phase-stable, 1.2 mJ, 1.5 cycle laser pulses at 2.1 &#181;m.</a> Opt Lett. 37 (23), 4973-4975 (2012).</li><li>Rothhardt, J., Demmler, S., H&#228;drich, S., Limpert, J., T&#252;nnermann, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Octave-spanning+OPCPA+system+delivering+CEP-stable+few-cycle+pulses+and+22+W+of+average+power+at+1+MHz+repetition+rate.">Octave-spanning OPCPA system delivering CEP-stable few-cycle pulses and 22 W of average power at 1 MHz repetition rate.</a> Opt express. 20 (10), 10870-10878 (2012).</li><li>Heckl, O. H., Nolte, S., Schrempel, F., Dausinger, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Ultrafast Thin-Disk Lasers.Ultrashort Pulse Laser Technology. 195, 93-115 (2016).</li><li>Zapata, L. E., 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=Cryogenic+Yb:YAG+composite-thin-disk+for+high+energy+and+average+power+amplifiers.">Cryogenic Yb:YAG composite-thin-disk for high energy and average power amplifiers.</a> Opt. Lett. 40 (11), 2610-2613 (2015).</li><li>Schulz, M., 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=Yb:YAG+Innoslab+amplifier:+efficient+high+repetition+rate+subpicosecond+pumping+system+for+optical+parametric+chirped+pulse+amplification.">Yb:YAG Innoslab amplifier: efficient high repetition rate subpicosecond pumping system for optical parametric chirped pulse amplification.</a> Opt Lett. 36 (13), 2456-2458 (2011).</li><li>Roeser, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Millijoule+pulse+energy+high+repetition+rate+femtosecond+fiber+chirped-pulse+amplification+system.">Millijoule pulse energy high repetition rate femtosecond fiber chirped-pulse amplification system.</a> Opt Lett. 32 (24), 3495-3497 (2007).</li><li>Russbueldt, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=400+W+Yb:YAG+Innoslab+fs-amplifier.">400 W Yb:YAG Innoslab fs-amplifier.</a> Opt Express. 17 (15), 12230-12245 (2009).</li><li>Baumgarten, C., 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=0.5+kHz+repetition+rate+picosecond+laser.">0.5 kHz repetition rate picosecond laser.</a> Opt Lett. 41 (14), 3339-3342 (2016).</li><li>Klingebiel, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=220mJ,+1+kHz+Picosecond+Regenerative+Thin-Disk+Amplifier.">220mJ, 1 kHz Picosecond Regenerative Thin-Disk Amplifier.</a> , (2015).</li><li>Nubbemeyer, T., 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=1+kW,+200+mJ+picosecond+thin-disk+laser+system.">1 kW, 200 mJ picosecond thin-disk laser system.</a> Opt Lett. 42 (7), 1381-1384 (2017).</li><li>Fattahi, H., 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=High-power,+1-ps,+all-Yb:YAG+thin-disk+regenerative+amplifier.">High-power, 1-ps, all-Yb:YAG thin-disk regenerative amplifier.</a> Opt Lett. 41 (6), 1126-1129 (2016).</li><li>Brons, 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=Energy+scaling+of+Kerr-lens+mode-locked+thin-disk+oscillators.">Energy scaling of Kerr-lens mode-locked thin-disk oscillators.</a> Opt Lett. 39 (22), 6442-6445 (2014).</li><li>Trebino, 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=Measuring+ultrashort+laser+pulses+in+the+time-frequency+domain+using+frequency-resolved+optical+gating.">Measuring ultrashort laser pulses in the time-frequency domain using frequency-resolved optical gating.</a> Rev Sci Instrum. 68 (9), 3277-3295 (1997).</li><li>Arisholm, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=General+numerical+methods+for+simulating+second-order+nonlinear+interactions+in+birefringent+media.">General numerical methods for simulating second-order nonlinear interactions in birefringent media.</a> J Opt Soc Am B. 14 (10), 2543-2549 (1997).</li><li>Zhang, D. X., Kong, Y. F., Zhang, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+parametric+properties+of+532-nm-pumped+beta-barium-borate+near+the+infrared+absorption+edge.">Optical parametric properties of 532-nm-pumped beta-barium-borate near the infrared absorption edge.</a> Opt Commun. 184 (5), 485-491 (2000).</li><li>Kato, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature-tuned+90o+phase-matching+properties+of+LiB3O5.">Temperature-tuned 90o phase-matching properties of LiB3O5.</a> IEEE J Quant Electron. 30 (12), 2950-2952 (1994).</li><li>Fattahi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Third-generation femtosecond technology. , (2016).</li></ol>

The authors have nothing to disclose.

The turn-key operation of the oscillator is achieved by the optimum heat management of the different components of the laser. The output of the oscillator is reproducible on a daily basis, with no need for extra alignment or optimization. In addition, the pulse-to-pulse energy stability and spatial pointing stability of the seed laser fulfills the preconditions to achieving the stable operation of the regenerative amplifier.
Other low-energy seed sources, such as fiber amplifiers, can be used to seed the amplifier. In this study, a 2 &#181;J Yb:YAG thin-disk KLM oscillator was used to assist the amplification of the regenerative amplifier by reducing the growth of the accumulated nonlinear phases, since the required number of round trips is reduced for higher-input seed energy. Additionally, the higher seed energy influences the amplification process and reduces the gain narrowing. The measured spectral bandwidth of the amplified pulses for different seed energies at a fixed pump power is shown in Figure 5c. Amplified spectral bandwidth decreases for lower seed energies because of gain narrowing. For 10 pJ seed energy, the laser operates in the period doubling, and it is not possible to reach stable operation, even by increasing the number of round trips. In addition to the careful optimization of the cooling systems and the power supply of the diodes, the operation of the regenerative amplifier at saturation plays a major role in the achieved stability of the amplifier.
The fundamental or second harmonic of the laser can be used to pump an OPCPA system. For SHG, the performances of an LBO and a BBO crystal were compared, as they offer a high nonlinear coefficient and damage threshold, in spite of the larger spatial walk-off and the limited available aperture in the case of BBO. As the nonlinear coefficient of BBO is almost twice that of the LBO, a shorter crystal is sufficient to reach the saturation limit for SHG (Figure 6a). Therefore, BBO is the more suitable choice, as the accumulated nonlinear phase is smaller28.
The pulse durations of the SH pulses are characterized experimentally at different conversion efficiencies. It was observed that at high conversion efficiencies, the SHG spectrum is broadened and a higher-order spectral phase appears (Figure 6). Therefore, case B, with the conversion efficiency of 70%, is chosen where the SH and the unconverted fundamental beams maintain excellent quality.

The generation of high-energy, few-cycle laser pulses at a high repetition rate is of great interest to applied fields, such as attosecond science1,2,3,4 and high-field physics5,6, which stand to directly benefit from the availability of such sources. OPCPA represents the most promising route to achieving high pulse energies and large amplification bandwidths that simultaneously support few-cycle pulses1. To date, OPCPA allows for ultra-broadband amplification, which generates few-cycle pulses7,8,9,10. However, a modified implementation of the OPCPA scheme, which uses short pump pulses on the picosecond scale, holds promise for making this approach scalable for even higher pulse energies and average powers in the few-cycle regime1,11,12. Due to the high pump intensity in short-pulse pumped OPCPA, the high single-pass gain allows for the use of very thin crystals to support large amplification bandwidths. Although the short-pulse pumped OPCPA has many advantages, the realizability of this approach is subject to the availability of lasers that are specially tailored for this purpose. Such pump lasers are required to deliver high-energy picosecond pulses with near-diffraction limited beam quality at repetition rates in the kHz to MHz range13,14,15.
The introduction of ytterbium-doped lasers at different geometries, capable of delivering picosecond laser pulses with high energy and high average power, are about to change the current state of the field1,13,14,15,16,17,18. Yb:YAG has good thermal conductivity and a long upper-state lifetime, and it can be pumped by cost-effective diode lasers. Its performance when used in thin-disk geometry is outstanding due to the efficient cooling of the gain medium to simultaneously scale the peak and average power. Moreover, the occurrence of self-focusing inside the gain medium during the amplification process is suppressed due to the slenderness of the thin-disk in comparison to other gain medium geometries, resulting in excellent temporal and spatial profiles of the amplified pulses. Combining this concept with CPA holds promise for generating picosecond pulses with hundreds of millijoules of energy and hundreds of watts of average power19,20.
The aim of this work is to demonstrate a turn-key Yb:YAG thin-disk regenerative amplifier with outstanding daily performance as a suitable source for pumping OPCPAs21. To achieve this goal, this study employs a Yb:YAG thin-disk oscillator22 with several microjoules of pulse energy to seed the amplifier to minimize the accumulated nonlinear phase during the amplification process. This protocol provides the recipe for building and operating the laser system, which is described elsewhere21. Details about component implementation and control software are presented, and the alignment process of the system is described.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Electrooptics</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fiber-Coupled Diode Laser Module</td>
			<td>Dilas Diodenlaser GmbH</td>
			<td>M1F8H12-940.5-500C-IS11.34</td>
			<td></td>
		</tr>
		<tr>
			<td>Fiber-Coupled Diode Laser Module</td>
			<td>Laserline GmbH</td>
			<td>LDM1000-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Power Supply for Diode Laser</td>
			<td>Delta Elektronika B.V.</td>
			<td>SM 15-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Power Supply for Diode Laser</td>
			<td>Delta Elektronika B.V.</td>
			<td>SM 35-45</td>
			<td></td>
		</tr>
		<tr>
			<td>Pulse Picker&#39;s Driver</td>
			<td>Bergmann Messger&#228;te Entwicklung KG&#160;</td>
			<td>N/A, customized</td>
			<td></td>
		</tr>
		<tr>
			<td>Pockels Cell&#39;s Driver</td>
			<td>Bergmann Messger&#228;te Entwicklung KG&#160;</td>
			<td>N/A, customized</td>
			<td></td>
		</tr>
		<tr>
			<td>Pulse Picker&#39;s Driver Power Supply</td>
			<td>Bergmann Messger&#228;te Entwicklung KG&#160;</td>
			<td>PCD8m7</td>
			<td></td>
		</tr>
		<tr>
			<td>Pockels Cell&#39;s Driver Power Supply</td>
			<td>Bergmann Messger&#228;te Entwicklung KG&#160;</td>
			<td>PCD8m7</td>
			<td></td>
		</tr>
		<tr>
			<td>Delay Generator PCI</td>
			<td>Bergmann Messger&#228;te Entwicklung KG&#160;</td>
			<td>BME_SG08p</td>
			<td></td>
		</tr>
		<tr>
			<td>Splitter Box</td>
			<td>Bergmann Messger&#228;te Entwicklung KG&#160;</td>
			<td>N/A, customized</td>
			<td></td>
		</tr>
		<tr>
			<td>Resonant Preamplifier</td>
			<td>Bergmann Messger&#228;te Entwicklung KG&#160;</td>
			<td>BME_P03</td>
			<td></td>
		</tr>
		<tr>
			<td>Pulse Picker&#39;s crystal</td>
			<td>Castech Inc.</td>
			<td>N/A, customized</td>
			<td>12*12*20 mm&#179;</td>
		</tr>
		<tr>
			<td>Pockels Cell&#39;s crystal</td>
			<td>Castech Inc.</td>
			<td>N/A, customized</td>
			<td>12*12*20 mm&#179;</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Optics</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Thin-disk</td>
			<td>TRUMPF Scientific Lasers&#160;</td>
			<td>N/A, customized</td>
			<td></td>
		</tr>
		<tr>
			<td>Thin-disk Head</td>
			<td>TRUMPF Scientific Lasers&#160;</td>
			<td>N/A, customized</td>
			<td></td>
		</tr>
		<tr>
			<td>Fiber</td>
			<td>Frank Optic Products GmbH</td>
			<td>N/A, customized</td>
			<td></td>
		</tr>
		<tr>
			<td>Fiber Objective</td>
			<td>Edmund Optics GmbH</td>
			<td>N/A, customized</td>
			<td></td>
		</tr>
		<tr>
			<td>Faraday Isolator</td>
			<td>Electro-Optics Technology, Inc</td>
			<td>EOT.189.12231</td>
			<td></td>
		</tr>
		<tr>
			<td>Faraday Rotator</td>
			<td>Electro-Optics Technology, Inc</td>
			<td>EOT.189.22040</td>
			<td></td>
		</tr>
		<tr>
			<td>Stretcher&#39;s Grating 1</td>
			<td>Horiba Jobin Yvon GmbH</td>
			<td>N/A, customized</td>
			<td>60*40*10 mm&#179;</td>
		</tr>
		<tr>
			<td>Stretcher&#39;s Grating 2</td>
			<td>Horiba Jobin Yvon GmbH</td>
			<td>N/A, customized</td>
			<td>350*190*50 mm&#179;</td>
		</tr>
		<tr>
			<td>Compressor&#39;s Grating 1</td>
			<td>Plymouth Grating Laboratory, Inc.</td>
			<td>N/A, customized</td>
			<td>40*40*16 mm&#179;</td>
		</tr>
		<tr>
			<td>Compressor&#39;s Grating 2</td>
			<td>Plymouth Grating Laboratory, Inc.</td>
			<td>N/A, customized</td>
			<td>300*100*50 mm&#179;</td>
		</tr>
		<tr>
			<td>HR Mirror, 1&#34; (1030nm), flat, 0&#176;</td>
			<td>Layertec GmbH</td>
			<td>108060</td>
			<td></td>
		</tr>
		<tr>
			<td>HR Mirror, 1&#34; (1030nm), flat, 0&#176;</td>
			<td>Laseroptik GmbH</td>
			<td>B-09965, S-04484</td>
			<td></td>
		</tr>
		<tr>
			<td>HR Mirror, 1&#34; (1030nm), flat, 45&#176;</td>
			<td>Layertec GmbH</td>
			<td>108063</td>
			<td></td>
		</tr>
		<tr>
			<td>HR Mirror, 1&#34; (1030nm), flat, 45&#176;</td>
			<td>Laseroptik GmbH</td>
			<td>B-09966, S-04484</td>
			<td></td>
		</tr>
		<tr>
			<td>HR Mirror, 1&#34; (1030nm), curved</td>
			<td>Layertec GmbH</td>
			<td>N/A, customized</td>
			<td>set</td>
		</tr>
		<tr>
			<td>HR Mirror, 2&#34; (1030nm), flat, 0&#176;</td>
			<td>Laseroptik GmbH</td>
			<td>B-09965, S-05474</td>
			<td></td>
		</tr>
		<tr>
			<td>HR Mirror, 2&#34; (1030nm), flat, 45&#176;</td>
			<td>Laseroptik GmbH</td>
			<td>B-09966, S-05474</td>
			<td></td>
		</tr>
		<tr>
			<td>Thin Film Polarizer (1030nm), 2&#34;</td>
			<td>Layertec GmbH</td>
			<td>103930</td>
			<td></td>
		</tr>
		<tr>
			<td>Waveplate L/2 (1030nm)</td>
			<td>Layertec GmbH</td>
			<td>106058</td>
			<td>&#216;=25mm</td>
		</tr>
		<tr>
			<td>Waveplate L/4 (1030nm)</td>
			<td>Layertec GmbH</td>
			<td>106060</td>
			<td>&#216;=25mm</td>
		</tr>
		<tr>
			<td>AR Window (1030nm), wedge</td>
			<td>Laseroptik GmbH</td>
			<td>B-00183-01, S-00988</td>
			<td>&#216;=38mm</td>
		</tr>
		<tr>
			<td>Output Coupler, 1&#34; (1030nm)</td>
			<td>Layertec GmbH</td>
			<td>N/A, customized</td>
			<td>PR = 88 %</td>
		</tr>
		<tr>
			<td>High-dispersion Mirror (1030nm)</td>
			<td>UltraFast Innovations GmbH</td>
			<td>N/A, customized</td>
			<td>GDD = -3000 fs&#178;</td>
		</tr>
		<tr>
			<td>Lens, 1&#34; (1030nm), Plano-Convex</td>
			<td>Thorlabs GmbH</td>
			<td>N/A, customized</td>
			<td>set</td>
		</tr>
		<tr>
			<td>Lens, 1&#34; (1030nm), Plano-Concave</td>
			<td>Thorlabs GmbH</td>
			<td>N/A, customized</td>
			<td>set</td>
		</tr>
		<tr>
			<td>Lens, 2&#34; (1030nm), Plano-Convex</td>
			<td>Thorlabs GmbH</td>
			<td>N/A, customized</td>
			<td>set</td>
		</tr>
		<tr>
			<td>Lens, 2&#34; (1030nm), Plano-Concave</td>
			<td>Thorlabs GmbH</td>
			<td>N/A, customized</td>
			<td>set</td>
		</tr>
		<tr>
			<td>HR Mirror, 1&#34; (515nm), flat, 0&#176;</td>
			<td>Layertec GmbH</td>
			<td>129784</td>
			<td></td>
		</tr>
		<tr>
			<td>HR Mirror, 1&#34; (515nm), flat, 0&#176;</td>
			<td>Eksma Optics</td>
			<td>042-0515-i0</td>
			<td></td>
		</tr>
		<tr>
			<td>HR Mirror, 1&#34; (515nm), flat, 45&#176;</td>
			<td>Layertec GmbH</td>
			<td>110924</td>
			<td></td>
		</tr>
		<tr>
			<td>HR Mirror, 1&#34; (515nm), flat, 45&#176;</td>
			<td>Eksma Optics</td>
			<td>042-0515</td>
			<td></td>
		</tr>
		<tr>
			<td>HR Mirror, 1&#34; (515nm), curved</td>
			<td>Layertec GmbH</td>
			<td>N/A, customized</td>
			<td>set</td>
		</tr>
		<tr>
			<td>HR Mirror, 1&#34; (515nm), curved</td>
			<td>Eksma Optics</td>
			<td>N/A, customized</td>
			<td>set</td>
		</tr>
		<tr>
			<td>HR Mirror, 2&#34; (515nm), flat, 0&#176;</td>
			<td>Eksma Optics</td>
			<td>045-0515-i0</td>
			<td></td>
		</tr>
		<tr>
			<td>HR Mirror, 2&#34; (515nm), flat, 45&#176;</td>
			<td>Eksma Optics</td>
			<td>045-0515</td>
			<td></td>
		</tr>
		<tr>
			<td>Thin Film Polarizer (515nm), 2&#34;</td>
			<td>Layertec GmbH</td>
			<td>112544</td>
			<td></td>
		</tr>
		<tr>
			<td>Waveplate L/2 (515nm)</td>
			<td>Layertec GmbH</td>
			<td>112546</td>
			<td>&#216;=25mm</td>
		</tr>
		<tr>
			<td>Lens, 1&#34; (515nm), Plano-Convex</td>
			<td>Thorlabs GmbH</td>
			<td>N/A, customized</td>
			<td>set</td>
		</tr>
		<tr>
			<td>Lens, 1&#34; (515nm), Plano-Concave</td>
			<td>Thorlabs GmbH</td>
			<td>N/A, customized</td>
			<td>set</td>
		</tr>
		<tr>
			<td>Kerr Medium</td>
			<td>Meller Optics, Inc.</td>
			<td>N/A, customized</td>
			<td>Sapphire, 1mm</td>
		</tr>
		<tr>
			<td>BBO Crystal</td>
			<td>Castech Inc.</td>
			<td>N/A, customized</td>
			<td>7*7*1.5 mm&#179;</td>
		</tr>
		<tr>
			<td>Harmonic Separator, 1&#34;, 45&#176;</td>
			<td>Eksma Optics</td>
			<td>042-5135</td>
			<td></td>
		</tr>
		<tr>
			<td>Harmonic Separator, 2&#34;, 45&#176;</td>
			<td>Eksma Optics</td>
			<td>045-5135</td>
			<td></td>
		</tr>
		<tr>
			<td>Silver Mirror, 1&#34;, flat</td>
			<td>Thorlabs GmbH</td>
			<td>PF10-03-P01</td>
			<td></td>
		</tr>
		<tr>
			<td>Silver Mirror, 1&#34;, curved</td>
			<td>Eksma Optics</td>
			<td>N/A, customized</td>
			<td>set</td>
		</tr>
		<tr>
			<td>Filter - Absorptive Neutral Density</td>
			<td>Thorlabs GmbH</td>
			<td>NE##A</td>
			<td>set</td>
		</tr>
		<tr>
			<td>Filter - Reflective Neutral Density</td>
			<td>Thorlabs GmbH</td>
			<td>ND##A</td>
			<td>set</td>
		</tr>
		<tr>
			<td>Filter - Round Continuously Variable</td>
			<td>Thorlabs GmbH</td>
			<td>NDC-50C-4M</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter - Edgepass Filter (Longpass)</td>
			<td>Thorlabs GmbH</td>
			<td>FEL####</td>
			<td>set</td>
		</tr>
		<tr>
			<td>Filter - Edgepass Filter (Shortpass)</td>
			<td>Thorlabs GmbH</td>
			<td>FES####</td>
			<td>set</td>
		</tr>
		<tr>
			<td>Wedge</td>
			<td>Thorlabs GmbH</td>
			<td>N/A, customized</td>
			<td>set</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Optomechanics &#38; Motion</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mirror Mount 1&#34; (small)</td>
			<td>S. Maier GmbH</td>
			<td>S1M4-##-1&#8221;</td>
			<td></td>
		</tr>
		<tr>
			<td>Mirror Mount 1&#34; (large)</td>
			<td>S. Maier GmbH</td>
			<td>S3-##</td>
			<td></td>
		</tr>
		<tr>
			<td>Mirror Mount 1&#34;</td>
			<td>TRUMPF Scientific Lasers&#160;</td>
			<td>1&#34; adjustable&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Mirror Mount 2&#34;</td>
			<td>S. Maier GmbH</td>
			<td>S4-##</td>
			<td></td>
		</tr>
		<tr>
			<td>Mirror Mount 2&#34;</td>
			<td>TRUMPF Scientific Lasers&#160;</td>
			<td>2&#34; adjustable&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotation Mount 1&#8221;</td>
			<td>S. Maier GmbH</td>
			<td>D25</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotation Mount 1&#8221;</td>
			<td>Thorlabs GmbH</td>
			<td>RSP1/M</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotation Mount 2&#8221;</td>
			<td>Thorlabs GmbH</td>
			<td>RSP2/M</td>
			<td></td>
		</tr>
		<tr>
			<td>Precision Rotation Stage</td>
			<td>Newport Corporation</td>
			<td>M-UTR120</td>
			<td></td>
		</tr>
		<tr>
			<td>Four-Axis Diffraction Grating Mount</td>
			<td>Newport Corporation</td>
			<td>DGM-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Translation Stage</td>
			<td>OptoSigma Corporation</td>
			<td>TADC-651SR25-M6</td>
			<td></td>
		</tr>
		<tr>
			<td>Pockels cell stage</td>
			<td>Newport Corporation</td>
			<td>9082-M</td>
			<td></td>
		</tr>
		<tr>
			<td>Pockels Cell Holder</td>
			<td>Home-made</td>
			<td>N/A, customized</td>
			<td></td>
		</tr>
		<tr>
			<td>Picomotor Controller/Driver Kit</td>
			<td>Newport Corporation</td>
			<td>8742-12-KIT</td>
			<td></td>
		</tr>
		<tr>
			<td>Picomotor Piezo Linear Actuators</td>
			<td>Newport Corporation</td>
			<td>8301NF</td>
			<td></td>
		</tr>
		<tr>
			<td>Picomotor Rotation Mount</td>
			<td>Newport Corporation</td>
			<td>8401-M</td>
			<td></td>
		</tr>
		<tr>
			<td>Hand Control Pad</td>
			<td>Newport Corporation</td>
			<td>8758</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Light Analysis</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Beam Profiling Camera</td>
			<td>Ophir Optronics Solutions Ltd</td>
			<td>SP620</td>
			<td></td>
		</tr>
		<tr>
			<td>Beam Profiling Camera</td>
			<td>DataRay Inc.</td>
			<td>WCD-UCD23</td>
			<td></td>
		</tr>
		<tr>
			<td>Photodiodes (solw)</td>
			<td>Thorlabs GmbH</td>
			<td>DET10A/M</td>
			<td></td>
		</tr>
		<tr>
			<td>Photodiodes (fast)</td>
			<td>Alphalas GmbH</td>
			<td>UPD-200-SP</td>
			<td></td>
		</tr>
		<tr>
			<td>Thin-disk Camera</td>
			<td>Imaging Development Systems GmbH</td>
			<td>UI-2220SE-M-GL</td>
			<td></td>
		</tr>
		<tr>
			<td>Oscilloscope</td>
			<td>Tektronix GmbH</td>
			<td>DPO5204</td>
			<td></td>
		</tr>
		<tr>
			<td>Oscilloscope</td>
			<td>Teledyne LeCroy GmbH</td>
			<td>SDA 760Zi-A</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrometer</td>
			<td>Avantes</td>
			<td>AvaSpec-ULS3648-USB2</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrometer</td>
			<td>Ocean Optics, Inc</td>
			<td>HR4C1769</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrometer</td>
			<td>Ocean Optics, Inc</td>
			<td>HR4C3762</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrometer</td>
			<td>Ocean Optics, Inc</td>
			<td>HR4D464</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrometer</td>
			<td>Ocean Optics, Inc</td>
			<td>HR4D466</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser Thermal Power Sensor</td>
			<td>Ophir Optronics Solutions Ltd</td>
			<td>L50(150)A-PF-35</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser Thermal Power Sensor</td>
			<td>Ophir Optronics Solutions Ltd</td>
			<td>FL500A</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser Thermal Power Sensor</td>
			<td>Ophir Optronics Solutions Ltd</td>
			<td>3A-P-V1</td>
			<td></td>
		</tr>
		<tr>
			<td>Power and Energy Meter</td>
			<td>Ophir Optronics Solutions Ltd</td>
			<td>Vega</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Systems</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Laser Beam Stabilization System</td>
			<td>TEM-Messtechnik GmbH</td>
			<td>Aligna</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser M&#178; Measuring System</td>
			<td>Ophir Optronics Solutions Ltd</td>
			<td>M&#178;-200s</td>
			<td></td>
		</tr>
		<tr>
			<td>FROG</td>
			<td>Home-made</td>
			<td>N/A, customized</td>
			<td></td>
		</tr>
		<tr>
			<td>XFROG</td>
			<td>Home-made</td>
			<td>N/A, customized</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Miscellaneous</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cooling Chiller</td>
			<td>H.I.B Systemtechnik GmbH</td>
			<td>6HE-000800-W-W-R23-2-DI</td>
			<td></td>
		</tr>
		<tr>
			<td>Cooling Chiller</td>
			<td>Termotek GmbH</td>
			<td>P201</td>
			<td></td>
		</tr>
		<tr>
			<td>Cooling Chiller</td>
			<td>Termotek GmbH</td>
			<td>P208</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser Safety Goggles</td>
			<td>Protect - Laserschutz GmbH</td>
			<td>BGU 10-0165-G-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Infra-red Viewer</td>
			<td>FJW Optical Systems</td>
			<td>84499A</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser Viewing Card</td>
			<td>Thorlabs GmbH</td>
			<td>VRC4</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser Viewing Card</td>
			<td>Thorlabs GmbH</td>
			<td>VRC5</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser Viewing Card</td>
			<td>Laser Components GmbH</td>
			<td>LDT-1064 BG</td>
			<td></td>
		</tr>
		<tr>
			<td>Flowmeter</td>
			<td>KOBOLD Messring GmbH</td>
			<td>DTK-1250G2C34P</td>
			<td></td>
		</tr>
		<tr>
			<td>Pressure Gauge</td>
			<td>KOBOLD Messring GmbH</td>
			<td>EN 837-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Temperature Sensor</td>
			<td>KOBOLD Messring GmbH</td>
			<td>TDA-15H* ***P3M</td>
			<td></td>
		</tr>
		<tr>
			<td>WinLase Software</td>
			<td>Dr. C. Horvath &#38; Dr. F. Loesel</td>
			<td>WinLase Version 2.1 pro.</td>
			<td>Laser Cavity Software</td>
		</tr>
	</tbody>
</table>

20 mj, 1 ps yb:yag thin-disk regenerative amplifier

This is a report on a 100 W, 20 mJ, 1 ps Yb:YAG thin-disk regenerative amplifier. A homemade Yb:YAG thin-disk, Kerr-lens mode-locked oscillator with turn-key performance and microjoule-level pulse energy is used to seed the regenerative chirped-pulse amplifier. The amplifier is placed in airtight housing. It operates at room temperature and exhibits stable operation at a 5 kHz repetition rate, with a pulse-to-pulse stability less than 1%. By employing a 1.5 mm-thick beta barium borate crystal, the frequency of the laser output is doubled to 515 nm, with an average power of 70 W, which corresponds to an optical-to-optical efficiency of 70%. This superior performance makes the system an attractive pump source for optical parametric chirped-pulse amplifiers in the near-infrared and mid-infrared spectral range. Combining the turn-key performance and the superior stability of the regenerative amplifier, the system facilitates the generation of a broadband, CEP-stable seed. Providing the seed and pump of the optical parametric chirped-pulse amplification (OPCPA) from one laser source eliminates the demand of active temporal synchronization between these pulses. This work presents a detailed guide to set up and operate a Yb:YAG thin-disk regenerative amplifier, based on chirped-pulse amplification (CPA), as a pump source for an optical parametric chirped-pulse amplifier.

The oscillator delivers 350 fs, 2 &#181;J, 25-W pulses at 11 MHz repetition rate, with a pulse-to-pulse stability of 1% (rms) and beam-pointing fluctuations of less than 0.6% over 1 h of measurement (Figure 4).
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/55717/55717fig4.jpg" /> 
Figure 4: Yb:YAG thin-disk, Kerr-lens mode-locked oscillator. (a) The spectrum (red), the retrieved temporal intensity profile (blue), and the spatial profile (inset) of the oscillator pulses. (b) Measured and retrieved SHG-FROG spectrograph of the oscillator. This figure has been modified from Fattahi et al., with permission from reference21. <a href="http://cloudflare.jove.com/files/ftp_upload/55717/55717fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The seed pulses are amplified in the regenerative amplifier to 125 W while being pumped with a CW fiber-coupled diode at a wavelength of 940 nm at 280 W, corresponding to an optical-to optical efficiency of 47%. The pulse-to-pulse stability of the amplifier is less than 1%, and the amplifier exhibits excellent long-term stability after 10 h of continuous operation. The amplified beam has an excellent spatial profile, with a M2 of 1 (M2x = 1.08 and M2y = 1.07) and an excellent temporal profile after compression to 1 ps (at FWHM) (Figure 5).
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/55717/55717fig5.jpg" /> 
Figure 5: Characterization of the regenerative amplifier output and the gain-narrowing effect. (a) The stability of the regenerative amplifier average power after 10 h of continuous operation. Inset: (a-1) Normalized power to its mean value in a time window of 0.5 h; (a-2) Output beam profile of the regenerative amplifier. (b) Amplifier output spectrum (green) and the retrieved temporal intensity (blue) of the laser pulses at 100 W average power after the grating compressor. (c) Seed energy versus spectral bandwidth (FWHM) of the amplifier output and the required round trips for the same output average power at 300 W of pump power. This figure has been modified from Fattahi et al., with permission from reference21. <a href="http://cloudflare.jove.com/files/ftp_upload/55717/55717fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The SHG was analyzed using the SISYFOS code25. Two different crystals with the following parameters were considered: 1) a type-I, 6 mm-thick lithium triborate (LBO), with a phase-matching angle of 13.7&#176; and a nonlinear coefficient of 0.819 pm/V, and 2) a type-I, 3 mm-thick BBO with a phase-matching angle of 23.4&#176; and a nonlinear coefficient of 2 pm/V26,27. 1-ps, 20-mJ pulses at 1,030 nm and a peak intensity of 100 GW/cm2 were considered as the input of the simulation. The simulation results showed that the BBO performance was superior to that of the LBO for SHG (Figure 6).
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/55717/55717fig6.jpg" /> 
Figure 6: Second harmonic generation. (a) Simulated SHG energy for a 6 mm-thick LBO crystal and a 3 mm-thick BBO crystal. (b) Experimental SHG efficiency versus input pump peak intensity in a 1.5 mm-thick BBO crystal using 0.5 mJ (black) and 20 mJ (green) of the amplifier output. (c) The retrieved spectral intensity and (d) the group delay of XFROG measurements for different SHG efficiencies corresponding to points A, B, and C in (b). This figure has been modified from Fattahi et al., with permission from reference21. <a href="http://cloudflare.jove.com/files/ftp_upload/55717/55717fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about 20 mJ, 1 ps Yb:YAG Thin-disk Regenerative Amplifier at JoVE.com

20 mJ, 1 ps Yb:YAG Thin-disk Regenerative Amplifier

A protocol for the operation of a high-energy, high-power optical parametric chirped pulse amplifier pump source based on an Yb:YAG thin-disk regenerative amplifier is presented here.

This is a report on a 100 W, 20 mJ, 1 ps Yb:YAG thin-disk regenerative amplifier. A homemade Yb:YAG thin-disk, Kerr-lens mode-locked oscillator with ...

20-mj-1-ps-ybyag-thin-disk-regenerative-amplifier

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11.4K Views.  Ludwig Maximilian University of Munich. A protocol for the operation of a high-energy, high-power optical parametric chirped pulse amplifier pump source based on an Yb:YAG thin-disk regenerative amplifier is presented here.

Video: 20 mJ, 1 ps Yb:YAG Thin-disk Regenerative Amplifier

Microfluidic Picoliter Bioreactor for Microbial Single-cell Analysis: Fabrication, System Setup, and Operation

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described in detail. The PLBR can be utilized for the analysis of single bacteria and microcolonies to investigate biotechnological and microbiological related questions concerning, e.g.&#160;cell growth, morphology, stress response, and metabolite or protein production on single-cell level. The device features continuous media flow enabling constant environmental conditions for perturbation studies, but in addition allows fast medium changes as well as oscillating conditions to mimic any desired environmental situation. To fabricate the single use devices, a silicon wafer containing sub micrometer sized SU-8 structures served as the replication mold for rapid polydimethylsiloxane casting. Chips were cut, assembled, connected, and set up onto a high resolution and fully automated microscope suited for time-lapse imaging, a powerful tool for spatio-temporal cell analysis. Here, the biotechnological platform organism Corynebacterium glutamicum&#160;was seeded into the PLBR and cell growth and intracellular fluorescence were followed over several hours unraveling time dependent population heterogeneity on single-cell level, not possible with conventional analysis methods such as flow cytometry. Besides insights into device fabrication, furthermore, the preparation of the preculture, loading, trapping of bacteria, and the PLBR cultivation of single cells and colonies is demonstrated. These devices will add a new dimension in microbiological research to analyze time dependent phenomena of single bacteria under tight environmental control. Due to the simple and relatively short fabrication process the technology can be easily adapted at any microfluidics lab and simply tailored towards specific needs.

In this protocol the fabrication, setup and basic operation of a microfluidic picoliter bioreactor (PLBR) for single-cell analysis of prokaryotic microorganisms is introduced. Industrially relevant microorganisms were analyzed as proof of principle allowing insights into growth rate, morphology, and phenotypic heterogeneity over certain time periods, hardly possible with conventional methods.

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described ...

Harmonic Nanoparticles for Regenerative Research

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation nanoparticles (HNPs). The latter are a new family of probes recently introduced for labeling biological samples for multi-photon imaging. HNPs are capable of doubling the frequency of excitation light by the nonlinear optical process of second harmonic generation with no restriction on the excitation wavelength.
Multi-photon based methodologies for hESC differentiation into cardiac clusters (maintained as long term air-liquid cultures) are presented in detail. In particular, evidence on how to maximize the intense second harmonic (SH) emission of isolated HNPs during 3D monitoring of beating cardiac tissue in 3D is shown. The analysis of the resulting images to retrieve 3D displacement patterns is also detailed.

Protocol details are provided for in vitro labeling human embryonic stem cells with second harmonic generating nanoparticles. Methodologies for hESC investigation by multi-photon microscopy and their differentiation into cardiac clusters are also presented.

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Neonatal Cardiac Scaffolds: Novel Matrices for Regenerative Studies

The only definitive therapy for end stage heart failure is orthotopic heart transplantation. Each year, it is estimated that more than 100,000 donor hearts are needed for cardiac transplantation procedures in the United States1-2. Due to the limited numbers of donors, only approximately 2,400 transplants are performed each year in the U.S.2. Numerous approaches, from cell therapy studies to implantation of mechanical assist devices, have been undertaken, either alone or in combination, in an attempt to coax the heart to repair itself or to rest the failing heart3. In spite of these efforts, ventricular assist devices are still largely used for the purpose of bridging to transplantation and the utility of cell therapies, while they hold some curative promise, is still limited to clinical trials. Additionally, direct xenotransplantation has been attempted but success has been limited due to immune rejection. Clearly, another strategy is required to produce additional organs for transplantation and, ideally, these organs would be autologous so as to avoid the complications associated with rejection and lifetime immunosuppression. Decellularization is a process of removing resident cells from tissues to expose the native extracellular matrix (ECM) or scaffold. Perfusion decellularization offers complete preservation of the three dimensional structure of the tissue, while leaving the bulk of the mechanical properties of the tissue intact4. These scaffolds can be utilized for repopulation with healthy cells to generate research models and, possibly, much needed organs for transplantation. We have exposed the scaffolds from neonatal mice (P3), known to retain remarkable cardiac regenerative capabilities,5-8 to detergent mediated decellularization and we repopulated these scaffolds with murine cardiac cells. These studies support the feasibility of engineering a neonatal heart construct. They further allow for the investigation as to whether the ECM of early postnatal hearts may harbor cues that will result in improved recellularization strategies.

In these studies, we provide methodology for novel, neonatal, murine cardiac scaffolds for use in regenerative studies.

The only definitive therapy for end stage heart failure is orthotopic heart transplantation.  Each year, it is estimated that more than 100,000 donor ...

Studying Soft-matter and Biological Systems over a Wide Length-scale from Nanometer and Micrometer Sizes at the Small-angle Neutron Diffractometer KWS-2

The KWS-2 SANS diffractometer is dedicated to the investigation of soft matter and biophysical systems covering a wide length scale, from nm to &#181;m. The instrument is optimized for the exploration of the wide momentum transfer Q range between 1x10-4 and 0.5 &#197;-1 by combining classical pinhole, focusing (with lenses), and time-of-flight (with chopper) methods, while simultaneously providing high-neutron intensities with an adjustable resolution. Because of its ability to adjust the intensity and the resolution within wide limits during the experiment, combined with the possibility to equip specific sample environments and ancillary devices, the KWS-2 shows a high versatility in addressing the broad range of structural and morphological studies in the field. Equilibrium structures can be studied in static measurements, while dynamic and kinetic processes can be investigated over time scales between minutes to tens of milliseconds with time-resolved approaches. Typical systems that are investigated with the KWS-2 cover the range from complex, hierarchical systems that exhibit multiple structural levels (e.g., gels, networks, or macro-aggregates) to small and poorly-scattering systems (e.g., single polymers or proteins in solution). The recent upgrade of the detection system, which enables the detection of count rates in the MHz range, opens new opportunities to study even very small biological morphologies in buffer solution with weak scattering signals close to the buffer scattering level at high Q.
In this paper, we provide a protocol to investigate samples with characteristic size levels spanning a wide length scale and exhibiting ordering in the mesoscale structure using KWS-2. We present in detail how to use the multiple working modes that are offered by the instrument and the level of performance that is achieved.

Here, we present a protocol to investigate soft matter and biophysical systems over a wide mesoscopic length scale, from nm to &#181;m that involves the use of the KWS-2 SANS diffractometer at high intensities and an adjustable resolution.

The KWS-2 SANS diffractometer is dedicated to the investigation of soft matter and biophysical systems covering a wide length scale, from nm to &#181;m. ...

3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous microscale cryogels (microcryogels), which can be loaded with a variety of cell types to form 3D microtissues. Herein, we present the protocol to fabricate versatile 3D microtissues and their applications in regenerative therapy and drug screening. Size and shape-controllable microcryogels can be fabricated on an array chip, which can be harvested off-chip as individual cell-loaded carriers for injectable regenerative therapy or be further assembled on-chip into 3D microtissue arrays for high-throughput drug screening. Due to the high elastic nature of these microscale cryogels, the 3D microtissues exhibit great injectability for minimally invasive cell therapy by protecting cells from mechanical shear force during injection. This ensures enhanced cell survival and therapeutic effect in the mouse limb ischemia model. Meanwhile, assembly of 3D microtissue arrays in a standard 384-multi-well format facilitates the use of common laboratory facilities and equipment, enabling high-throughput drug screening on this versatile 3D cell culture platform.

This protocol describes the fabrication of elastic 3D macroporous microcryogels by integrating microfabrication with cryogelation technology. Upon loading with cells, 3D microtissues are generated, which can be readily injected in vivo to facilitate regenerative therapy or assembled into arrays for in vitro high-throughput drug screening.

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous ...

Generic Protocol for Optimization of Heterologous Protein Production Using Automated Microbioreactor Technology

A core business in industrial biotechnology using microbial production cell factories is the iterative process of strain engineering and optimization of bioprocess conditions. One important aspect is the improvement of cultivation medium to provide an optimal environment for microbial formation of the product of interest. It is well accepted that the media composition can dramatically influence overall bioprocess performance. Nutrition medium optimization is known to improve recombinant protein production with microbial systems and thus, this is a rewarding step in bioprocess development. However, very often standard media recipes are taken from literature, since tailor-made design of the cultivation medium is a tedious task that demands microbioreactor technology for sufficient cultivation throughput, fast product analytics, as well as support by lab robotics to enable reliability in liquid handling steps. Furthermore, advanced mathematical methods are required for rationally analyzing measurement data and efficiently designing parallel experiments such as to achieve optimal information content.
The generic nature of the presented protocol allows for easy adaption to different lab equipment, other expression hosts, and target proteins of interest, as well as further bioprocess parameters. Moreover, other optimization objectives like protein production rate, specific yield, or product quality can be chosen to fit the scope of other optimization studies. The applied Kriging Toolbox (KriKit) is a general tool for Design of Experiments (DOE) that contributes to improved holistic bioprocess optimization. It also supports multi-objective optimization which can be important in optimizing both upstream and downstream processes.

This manuscript describes a generic approach for tailor-made design of microbial cultivation media. This is enabled by an iterative workflow combining Kriging-based experimental design and microbioreactor technology for sufficient cultivation throughput, which is supported by lab robotics to increase reliability and speed in liquid handling media preparation.

A core business in industrial biotechnology using microbial production cell factories is the iterative process of strain engineering and optimization of ...

Recombinant Collagen I Peptide Microcarriers for Cell Expansion and Their Potential Use As Cell Delivery System in a Bioreactor Model

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation purposes. The process to generate such tissues is complex, and includes an appropriate combination of specific cell types, scaffolds, and physical or biochemical stimuli to guide cell growth and differentiation. Microcarriers represent an appealing tool to expand cells in a three-dimensional (3D) microenvironment, since they provide higher surface-to volume ratios and mimic more closely the in vivo situation compared to traditional two-dimensional methods. The vascular system, supplying oxygen and nutrients to the cells and ensuring waste removal, constitutes an important building block when generating engineered tissues. In fact, most constructs fail after being implanted due to lacking vascular support. In this study, we present a protocol for endothelial cell expansion on recombinant collagen-based microcarriers under dynamic conditions in spinner flask and bioreactors, and we explain how to determine in this setting cell viability and functionality. In addition, we propose a method for cell delivery for vascularization purposes without additional detachment steps necessary. Furthermore, we provide a strategy to evaluate the cell vascularization potential in a perfusion bioreactor on a decellularized&#160;biological matrix. We believe that the use of the presented methods could lead to the development of new cell-based therapies for a large range of tissue engineering applications in the clinical practice.

We propose a cell expansion protocol on macroporous microcarriers and their use as delivery system in a perfusion bioreactor to seed a decellularized tissue matrix. We also include different techniques to determine cell proliferation and viability of cells cultured on microcarriers. Furthermore, we demonstrate functionality of cells after bioreactor cultures.

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation ...

Construction of a Multilayered Mesenchymal Stem Cell Sheet with a 3D Dynamic Culture System

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low cell retention and poor survival within the target zone. However, during the in vitro construction process, a solution for maintaining stem cell bioactivity and increasing the cell amount within the cell sheet is urgently needed. Here, this protocol presents a method for constructing a multilayered cell sheet with favorable stem cell bioactivity and optimal operability. Decellularized porcine pericardium (DPP) is prepared by phospholipase A2 (PLA2) decellularization method as the cell sheet scaffold, and rat bone marrow mesenchymal stem cells (BMSCs) are isolated and expanded as the seeded cells. The temporary multilayered cell sheet structure is constructed by using RAD16-I peptide hydrogel. Finally, the cell sheet is cultured with a dynamic perfusion system to stabilize the three-dimensional (3D) structure, and the cell sheet could be obtained following a 48-hour culture in vitro. This protocol provides an efficient and feasible method for constructing a multilayered stem cell sheet, and the cell sheet could be developed as a favorable stem cell therapy product in the future.

This article provides an efficient and feasible method for constructing multilayered stem cell sheets with favorable stem cell property.

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low ...

Fabrication of the Composite Regenerative Peripheral Nerve Interface (C-RPNI) in the Adult Rat

Recent advances in neuroprosthetics have enabled those living with extremity loss to reproduce many functions native to the absent extremity, and this is often accomplished through integration with the peripheral nervous system. Unfortunately, methods currently employed are often associated with significant tissue damage which prevents prolonged use. Additionally, these devices often lack any meaningful degree of sensory feedback as their complex construction dampens any vibrations or other sensations a user may have previously depended on when using more simple prosthetics. The composite regenerative peripheral nerve interface (C-RPNI) was developed as a stable, biologic construct with the ability to amplify efferent motor nerve signals while providing simultaneous afferent sensory feedback. The C-RPNI consists of a segment of free dermal and muscle graft secured around a target mixed sensorimotor nerve, with preferential motor nerve reinnervation of the muscle graft and sensory nerve reinnervation of the dermal graft. In rats, this construct has demonstrated the generation of compound muscle action potentials (CMAPs), amplifying the target nerve&#39;s signal from the micro- to milli-volt level, with signal to noise ratios averaging approximately 30-50. Stimulation of the dermal component of the construct generates compound sensory nerve action potentials (CSNAPs) at the proximal nerve. As such, this construct has promising future utility towards the realization of the ideal, intuitive prosthetic.

Fabrication of the Composite Regenerative Peripheral Nerve Interface C-RPNI in the Adult Rat

The following manuscript describes a novel method for developing a biologic, closed loop neural feedback system termed the composite regenerative peripheral nerve interface (C-RPNI). This construct has the ability to integrate with peripheral nerves to amplify efferent motor signals while simultaneously providing afferent sensory feedback.

Recent advances in neuroprosthetics have enabled those living with extremity loss to reproduce many functions native to the absent extremity, and this is ...