We thank Dr. S. Tanaka at Tokyo Institute of Technology for time-resolved IR vibrational spectroscopy measurements and Prof. M. Hara and Dr. K. Matsuo at Nagoya University for XRD measurements. We also thanks Prof. S. Yamaguchi at Nagoya University, Prof. R. Herges at Kiel University and Prof. R. J. D. Miller at the Max Planck Institute for the Structure and Dynamics of Matter for valuable discussion.
This work is supported by the Japanese Science Technology (JST), PRESTO, for funding the projects "Molecular technology and creation of new functions" (Grant Number of JPMJPR13KD, JPMJPR12K5, and JPMJPR16P6) and "Chemical conversion of light energy". This work is also partially supported by JSPS Grant Numbers JP15H02103, JP17K17893, JP15H05482, JP17H05258, JP26107004, and JP17H06375.

1.Time-Resolved Infrared Vibrational Spectroscopy
<ol>
	<li>Sample preparation
	<ol>
		<li>Solution: Dissolve the &#960;-extended cyclooctatetraene (&#960;-COT) molecules into dichloromethane with proper concentration (1 mmol/L).</li>
		<li>LC phase: Melt the &#960;-COT powder on a calcium fluoride (CaF2) substrate using hot plate at the temperature of 100 &#176;C. Cool the sample at a room temperature. 
		Note: We need to choose a material (CaF2 or barium fluoride (BaF2)) that is transparent in mid-IR range.</li>
	</ol>
	</li>
	<li>Apparatus setting-up
	<ol>
		<li>Switch on the titanium sapphire (Ti:sapphire) laser and the chirped pulse amplifier. Thermally stabilize them for several hours.</li>
		<li>Make sure the alignments are correct. Check the power and stability of the ultraviolet (UV) pump and the mid-IR probe and re-align the optical path if necessary. The optical setup of the time-resolved infrared spectroscopy is provided in Figure 5.</li>
		<li>Cool the HgCdTe IR detector array using liquid nitrogen. Make sure that the spectrometer is properly located so that reasonable amount of light is detected in the range of interest. Calibrate the spectrometer using absorption spectra of well-known materials such as polystyrene or polyethylene terephthalate.</li>
		<li>Mount a sample which shows large photo-induced transient response (Si wafer (1 mm) or Re(bpy)(CO)3Cl/CH3CN solution) on the sample holder. Locate the pump-probe delay to a positive value and optimize the amount of the transient signal by stirring the pump beam to ensure the pump-probe overlap.</li>
		<li>Find the time origin setting by taking long-range scan on the pump-probe delay using the home-built program (Figure 6). Check the position where the transient signal start to emerge.</li>
		<li>Check the dynamics of symmetric and anti-symmetric vibration of CO stretching in Re(bpy)(CO)3Cl whose dipole moments are orthogonal. Note that both should show exactly same dynamics when the magic angle condition is properly met.</li>
	</ol>
	</li>
	<li>Measurement and data acquisition
	<ol>
		<li>Solution: Mount the home-built flow cell. Setup the bubbling device with inert gas (nitrogen (N2) or Argon (Ar)) if necessary. LC phase: Mount the spin-coated &#960;-COT sample with the substrate on the motorized stage to continuously move the laser spots on the sample to minimize the laser-induced damage.</li>
		<li>Doublecheck the time zero position with the sample.</li>
		<li>Set the scan range of the pump-probe delay properly (start, end, and step).</li>
		<li>Choose a directory to save data.</li>
		<li>Start the data collection with the home-built program. 
		NOTE: The data are recorded automatically in the directory.</li>
	</ol>
	</li>
</ol>
2. Time-Resolved Electron Diffraction
<ol>
	<li>Fabrication of sample substrate
	<ol>
		<li>Purchase a silicon (001) wafer (200 &#181;m thick), both sides of which are pre-covered with 30-nm-thick silicon-rich silicon nitride (Si3N4, or simply SiN) film (Figure 11A). Cut the SiN/Si/SiN wafer in square (15 &#215; 15 mm2).</li>
		<li>Irradiate with Ar cluster ion beams41 at the fluence of 2.5 &#215; 1016 ions/cm2 onto one of the sides of the SiN/Si/SiN wafer though a metal mask (Figure 12), which is sufficient to remove the 30-nm-thick SiN film (Figure 11B,C). 
		NOTE: An alternative method to remove SiN film is plasma etching or ion beams etching.</li>
		<li>Prepare potassium hydroxide (KOH)aqueous solution at a concentration of 28%.</li>
		<li>Put the wafer into KOH solution at a temperature of 60-70 &#176;C for 1-2 days (Figure 11D), which perform further etching of the Si wafer via isotropic chemical etching42. 
		NOTE: The etching rate for Si by KOH solution is much faster than that for SiN, so the SiN thin film remains as self-supporting membranes (Figure 11E).</li>
		<li>Clean the wafer with SiN membranes in deionized water and dry it with nitrogen gas.</li>
	</ol>
	</li>
	<li>Sample preparation
	<ol>
		<li>Dissolve the &#960;-COT molecules in chloroform at a concentration of 10 mg/mL.</li>
		<li>Program the spin-coater: accelerate to 2000 rpm in 5 s, keep the rotation for 30 s, and stop the rotation. Spin-coat the &#960;-COT solution onto the SiN membrane substrate as shown in Figure 11F. 
		NOTE: A proper wafer size for spin-coating must be more than 10 &#215; 10 mm2, since the surface tension sometimes interferes with spin-coating of materials on smaller wafers, for example, a SiN membrane grid for transmission electron microscopy.</li>
		<li>Put the sample coated on the SiN membrane substrate on a hotplate at a temperature of 100 &#176;C, melt it, and cool it gradually to room temperature (Figure 11G).</li>
	</ol>
	</li>
	<li>Measurements
	<ol>
		<li>Mount the sample on the sample holder with a screw and put the sample holder in the vacuum chamber (sample chamber).</li>
		<li>Seal the vacuum chamber with a lid and switch on a rotary pump to evacuate the chamber until a vacuum level of less than 1000 Pa. Then, switch on the molecular turbo pumps until the electron-gun chamber is at the vacuum level of ~10-6 Pa (typically for more than 12 h).</li>
		<li>Switch on the Ti:sapphire laser and the chirp pulse amplifier, and thermally stabilize them for more than 1 h. The experimental setup of the time-resolved electron diffraction is provided in Figure 9. Set the repetition rate to 500 Hz.</li>
		<li>Switch on the thriller of the charge-coupled device (CCD) camera and cool it to 10 &#176;C.</li>
		<li>Switch on the electrical power supply and adjust the voltage to 75 kV. 
		NOTE: The leak current of the power supply should not fluctuate out of the 0.1 &#181;A range.</li>
		<li>Special overlap. Open the laboratory-coded automatic program (Figure 10A) and set the exposure time (50 ms). Find the electron beam position with a pinhole equipped in the sample holder using the program by setting Start type to Z_overlap for the overlap of Z-axis and Y_overlap and pressing Start button.</li>
		<li>Set the electron beam at the pinhole position and align the pump laser with the reflected pump light by the pinhole.</li>
		<li>Measure the time-zero position with an inorganic material (Bi2Te3) on the sample holder using a laboratory-coded automatic program (Figure 10B) by setting Start type to Time-resolved and pressing Start button. For this process, adjust the pump fluence to 2 mJ/cm2.</li>
		<li>Insert the Faraday cup to the pass of the electron beam and measure the fluence of the electron beam with a laboratory-built picoammeter and adjust it by rotating the adjustable ND filter on the probe line. Adjust thefluence of the pump pulse by rotating the waveplate on the pump line.</li>
		<li>Move to the sample position and set the exposure time of the CCD camera. Obtain the electron diffraction image using the laboratory-coded automatic program (Figure 10B) by setting Start type to Single and pressing Start button.</li>
		<li>Switch on the Peltier element of the CCD camera and cool it down to the temperature of -20 &#176;C.</li>
		<li>Set the time-step and number of steps for the time-resolved measurements. Obtain the time-resolved electron diffraction images using the laboratory-coded automatic program (Figure 10B) by setting Start type to Time-resolved and pressing Start button.</li>
		<li>Obtain the time-resolved background image with the electron acceleration power supply switching off using the laboratory-coded automatic program (Figure 10B) by setting Start type to Time-resolved and pressing Start button.</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

The crucial step of the process during the time-resolved electron diffraction measurements is maintaining the high voltage (75 keV) without current fluctuation since the distance between the photocathode and anode plate is only ~10 mm. If the current fluctuates above the range of 0.1 &#181;A before or during the experiments, increase the acceleration voltage up to 90 keV to discharge and set it again to 75 keV. This conditioning process has to be done until the current fluctuates in the range of 0.1 &#181;A. The proper d...

Liquid crystals (LCs) have a variety of functions and are widely used in scientific and technological applications1,2,3,4,5,6. The behavior of LCs can be attributed to their orientational ordering as well as to the high mobility of their molecules. A molecular structure of LC materials is typically characterized by a mesogen core and long flexible carbon chains that ensure high mobility of the LC molecules. Under external stimuli7,8,9,10,11,12,13,14,15, such as light, electric fields, temperature changes, or mechanical pressure, small intra- and intermolecular motions of the LC molecules cause drastic structural reordering in the system, leading to its functional behavior. To understand the functions of LC materials, it is important to determine the molecular scale structure in the LC phase and identify the key motions of the molecular conformations and packing deformations.
X-ray diffraction (XRD) is commonly employed as a powerful tool for determining structures of LC materials16,17,18. However, the diffraction pattern originating from a functional stimuli-responsive core is often concealed by a broad halo pattern from the long carbon chains. An effective solution to this problem is provided by time-resolved diffraction analysis, which enables direct observations of molecular dynamics using photoexcitation. This technique extracts structural information about the photoresponsive aromatic moiety using the differences between the diffraction patterns obtained before and after photoexcitation. These differences provide the means both to remove the background noise and to directly observe the structural changes of interest. Analyses of the differential diffraction patterns reveal the modulated signals from the photoactive moiety alone, thereby excluding the deleterious diffraction from the non-photoresponsive carbon chains. A description of this method of differential diffraction analysis is provided in Hada, M. et al19.
Time-resolved diffraction measurements can provide structural information about the atomic rearrangements that occur during the phase transition in materials20,21,22,23,24,25,26,27,28,29 and chemical reactions among molecules30,31,32,33,34. With these applications in mind, remarkable progress has been made in the development of ultrabright and ultrashort-pulsed X-ray35,36 and electron37,38,39,40 sources. However, time-resolved diffraction has only been applied to simple, isolated molecules or to single- or poly-crystals, in which highly ordered inorganic lattice or organic molecules produce well-resolved diffraction patterns providing structural information. In contrast, ultrafast structural analyses of more complex soft materials have been hampered because of their less ordered phases. In this study, we demonstrate the use of time-resolved electron diffraction as well as transient absorption spectroscopy and time-resolved infrared (IR) vibrational spectroscopy to characterize the structural dynamics of photoactive LC materials using this diffraction-extracted methodology19.

<table border="0" cellpadding="0" cellspacing="0" style="width:572px;" width="573">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Chirped pulse amplifier</td>
			<td style="width:71px;">Spectra Physics Inc.</td>
			<td style="width:119px;">Spitfire ACE</td>
			<td style="width:167px;">For time-resolved IR vibration spectroscopy</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Chirped pulse amplifier&#160;</td>
			<td style="width:71px;">Spectra Physics Inc.</td>
			<td style="width:119px;">Spitfire XP</td>
			<td style="width:167px;">For time-resolved electron diffractometry</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Femtosecond laser</td>
			<td style="width:71px;">Spectra Physics Inc.</td>
			<td style="width:119px;">Tsunami</td>
			<td style="width:167px;">For time-resolved IR vibration spectroscopy</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Femtosecond laser</td>
			<td style="width:71px;">Spectra Physics Inc.</td>
			<td style="width:119px;">Tsunami</td>
			<td style="width:167px;">For time-resolved electron diffractometry</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Optical parametric amplifier</td>
			<td style="width:71px;">Light Conversion Ltd.</td>
			<td style="width:119px;">TOPAS prime</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:216px;">64-channel mercury cadmium tellurium IR detector array</td>
			<td style="width:71px;">Infrared Systems Development Corporation</td>
			<td style="width:119px;">FPAS-6416-D</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">FT-IR spectrometer</td>
			<td style="width:71px;">Shimadzu Corporation</td>
			<td style="width:119px;">IR Prestige-21</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">High voltage supply</td>
			<td style="width:71px;">Matsusada precision</td>
			<td style="width:119px;">HER-100N0.1</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Rotary pump</td>
			<td style="width:71px;">Edwards</td>
			<td style="width:119px;">RV12</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">Molecular turbo pumps</td>
			<td style="width:71px;">Agilent Technologies Japan, Ltd.</td>
			<td style="width:119px;">Twis Torr 304FS</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Vacuum gauges</td>
			<td style="width:71px;">Pfeiffer vacuum systems gmbh</td>
			<td style="width:119px;">PKR251</td>
			<td style="width:167px;">For ICF70 flange</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Vacuum monitors</td>
			<td style="width:71px;">Pfeiffer vacuum systems gmbh</td>
			<td style="width:119px;">TPG261</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Fiber coupled CCD camera</td>
			<td style="width:71px;">Andor Technology Ltd.</td>
			<td style="width:119px;">iKon-L HF</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">BaF2 and CaF2 substrates</td>
			<td style="width:71px;">Pier optics</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Thickness 3 mm</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">AgGaS2 crystal</td>
			<td style="width:71px;">Phototechnica Corporation</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Custom-order</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">BBO crystals</td>
			<td style="width:71px;">Tokyo Instruments, Inc.</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">SHG &#952;=29.2 deg 
			THG &#952;=44.3 deg</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">calcite crystals</td>
			<td style="width:71px;">Tokyo Instruments, Inc.</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Thickness 1mm</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">Optical mirrors</td>
			<td style="width:71px;">Thorlabs</td>
			<td style="width:119px;">PF10-03-F01 
			PF10-03-M01 
			UM10-45A</td>
			<td style="width:167px;">Al coat mirrors 
			Au coat mirrors 
			Ultrafast mirrors</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Optical mirrors</td>
			<td style="width:71px;">HIKARI,Inc.</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Broadband mirrors</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">Dichroic mirrors</td>
			<td style="width:71px;">HIKARI,Inc.</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Custom-order 
			Reflection: 266 nm 
			Transmission: 400, 800 nm</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Optical chopper</td>
			<td style="width:71px;">Newport Corporation</td>
			<td style="width:119px;">3501 optical chopper</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Optical shutters</td>
			<td style="width:71px;">Thorlabs Inc.</td>
			<td style="width:119px;">SH05/M 
			SC10</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Optical shutters</td>
			<td style="width:71px;">SURUGA SEIKI CO.,LTD.</td>
			<td style="width:119px;">F116-1</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Beam splitters</td>
			<td style="width:71px;">Thorlabs Inc.</td>
			<td style="width:119px;">BSS11R</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Fused-silica lenses</td>
			<td style="width:71px;">Thorlabs Inc.</td>
			<td style="width:119px;">LA4663 
			LA4184</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">BaF2 lens</td>
			<td style="width:71px;">Thorlabs Inc.</td>
			<td style="width:119px;">LA0606-E</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="95">
			<td height="95" style="height:95px;width:216px;">Polarized mirrors</td>
			<td style="width:71px;">Sigmakoki Co.,Ltd</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Custom-order 
			Designed for 800 nm 
			Reflection: s-polarized light 
			Transmission : p-polarized light</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Half waveplate</td>
			<td style="width:71px;">Thorlabs Inc.</td>
			<td style="width:119px;">WPH05M-808</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Mirror mounts</td>
			<td style="width:71px;">Thorlabs Inc.</td>
			<td style="width:119px;">POLARIS-K1 
			KM100</td>
			<td style="width:167px;">Kinematic mirror mounts</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Mirror mounts</td>
			<td style="width:71px;">Sigmakoki Co.,Ltd</td>
			<td style="width:119px;">MHAN-30M 
			MHAN-30S</td>
			<td style="width:167px;">Gimbal mirror mounts</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Mirror mounts</td>
			<td style="width:71px;">Newport Corporation</td>
			<td style="width:119px;">ACG-3K-NL</td>
			<td style="width:167px;">Gimbal mirror mounts</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Variable ND filters</td>
			<td style="width:71px;">Thorlabs Inc.</td>
			<td style="width:119px;">NDC-25C-2M</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Beam splitter mounts</td>
			<td style="width:71px;">Thorlabs Inc.</td>
			<td style="width:119px;">KM100S</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Lens mounts</td>
			<td style="width:71px;">Thorlabs Inc.</td>
			<td style="width:119px;">LMR1/M</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Rotational mounts</td>
			<td style="width:71px;">Thorlabs Inc.</td>
			<td style="width:119px;">RSP1/M</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Retroreflector</td>
			<td style="width:71px;">Edmund Optics</td>
			<td style="width:119px;">63.5MM X 30&#34; EN-AL&#160;</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">spectrometers</td>
			<td style="width:71px;">ocean photonics</td>
			<td style="width:119px;">USB-4000</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Power meter</td>
			<td style="width:71px;">Ophir</td>
			<td style="width:119px;">30A-SH</td>
			<td style="width:167px;">Used for intensity monitor of CPA</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Power meter</td>
			<td style="width:71px;">Thorlabs Inc.</td>
			<td style="width:119px;">S120VC 
			PM100USB</td>
			<td style="width:167px;">Used for intensity measurements of pump pulse</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Photodiodes</td>
			<td style="width:71px;">Thorlabs Inc.</td>
			<td style="width:119px;">DET36A/M 
			DET25K/M</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DC power supply</td>
			<td style="width:71px;">TEXIO</td>
			<td style="width:119px;">PW18-1.8AQ</td>
			<td style="width:167px;">Used for magnetic lens</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Magnetic lens</td>
			<td style="width:71px;">Nissei ETC Co.,Ltd</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Custom-order</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Stages</td>
			<td style="width:71px;">Newport Corporation</td>
			<td style="width:119px;">M-MVN80V6 
			LTAHLPPV6</td>
			<td style="width:167px;">Used for magnetic lens</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Stage controller</td>
			<td style="width:71px;">Newport Corporation</td>
			<td style="width:119px;">SMC100</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Stages&#160;</td>
			<td style="width:71px;">Sigmakoki Co.,Ltd</td>
			<td style="width:119px;">SGSP20-35(X) 
			SGSP20-85(X)</td>
			<td style="width:167px;">Used for sample position</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Stages&#160;</td>
			<td style="width:71px;">Sigmakoki Co.,Ltd</td>
			<td style="width:119px;">SGSP26-200(X) 
			OSMS26-300(X)</td>
			<td style="width:167px;">Used for delay time generator</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Stage controller</td>
			<td style="width:71px;">Sigmakoki Co.,Ltd</td>
			<td style="width:119px;">SHOT-304GS</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Picoammeter</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Laboratory built</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">spin coater</td>
			<td style="width:71px;">MIKASA Co.,Ltd</td>
			<td style="width:119px;">1H-D7</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">hot plate</td>
			<td style="width:71px;">IKA&#174;&#160;</td>
			<td style="width:119px;">C-MAG HP7</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SiN wafer</td>
			<td style="width:71px;">Silson Ltd</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Custom-order</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">KOH aqueous solution (50%)</td>
			<td style="width:71px;">Hiroshima Wako Co.,Ltd.</td>
			<td style="width:119px;">168-20455</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Chloroform</td>
			<td style="width:71px;">Hiroshima Wako Co.,Ltd.</td>
			<td style="width:119px;">038-18495</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Dichloromethane</td>
			<td style="width:71px;">Hiroshima Wako Co.,Ltd.</td>
			<td style="width:119px;">132-02456</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Personal computers for the controlling programs</td>
			<td style="width:71px;">Epson Corporate</td>
			<td style="width:119px;">Endeavor MR7300E-L</td>
			<td style="width:167px;">32-bit operation system</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">Program for the control the equipment</td>
			<td style="width:71px;">National Instruments Corporation</td>
			<td style="width:119px;">Labview2016</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Program for the data analysis</td>
			<td style="width:71px;">The MathWorks, Inc.</td>
			<td style="width:119px;">Matlab2015b</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

novel techniques for observing structural dynamics of photoresponsive liquid crystals

We discuss in this article the experimental measurements of the molecules in liquid crystal (LC) phase using the time-resolved infrared (IR) vibrational spectroscopy and time-resolved electron diffraction. Liquid crystal phase is an important state of matter that exists between the solid and liquid phases and it is common in natural systems as well as in organic electronics. Liquid crystals are orientationally ordered but loosely packed, and therefore, the internal conformations and alignments of the molecular components of LCs can be modified by external stimuli. Although advanced time-resolved diffraction techniques have revealed picosecond-scale molecular dynamics of single crystals and polycrystals, direct observations of packing structures and ultrafast dynamics of soft materials have been hampered by blurry diffraction patterns. Here, we report time-resolved IR vibrational spectroscopy and electron diffractometry to acquire ultrafast snapshots of a columnar LC material bearing a photoactive core moiety. Differential-detection analyses of the combination of time-resolved IR vibrational spectroscopy and electron diffraction are powerful tools for characterizing structures and photoinduced dynamics of soft materials.

We chose a saddle-shaped &#960;-COT skeleton43,44 as a photoactive core unit of the LC molecule, because it forms a well-defined columnar stacking structure and because the central eight-membered COT ring is expected to show a photoinduced conformational change into a flat form owing to the excited-state aromaticity19,45. Synthetic process of this material is provided in p...

Watch this Scientific Journal Video about Novel Techniques for Observing Structural Dynamics of Photoresponsive Liquid Crystals at JoVE.com

Novel Techniques for Observing Structural Dynamics of Photoresponsive Liquid Crystals

Here, we present the protocols of differential-detection analyses of time-resolved infrared vibrational spectroscopy and electron diffraction which enable observations of the deformations of local structures around photoexcited molecules in a columnar liquid crystal, giving an atomic perspective on the relationship between the structure and the dynamics of this photoactive material.

We discuss in this article the experimental measurements of the molecules in liquid crystal (LC) phase using the time-resolved infrared (IR) vibrational ...

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Chemistry

8.7K Views.  Okayama University. Here, we present the protocols of differential-detection analyses of time-resolved infrared vibrational spectroscopy and electron diffraction which enable observations of the deformations of local structures around photoexcited molecules in a columnar liquid crystal, giving an atomic perspective on the relationship between the structure and the dynamics of this photoactive material.

Video: Novel Techniques for Observing Structural Dynamics of Photoresponsive Liquid Crystals

Production of Disulfide-stabilized Transmembrane Peptide Complexes for Structural Studies

Physical interactions among the lipid-embedded alpha-helical domains of membrane proteins play a crucial role in folding and assembly of membrane protein complexes and in dynamic processes such as transmembrane (TM) signaling and regulation of cell-surface protein levels. Understanding the structural features driving the association of particular sequences requires sophisticated biophysical and biochemical analyses of TM peptide complexes. However, the extreme hydrophobicity of TM domains makes them very difficult to manipulate using standard peptide chemistry techniques, and production of suitable study material often proves prohibitively challenging. Identifying conditions under which peptides can adopt stable helical conformations and form complexes spontaneously adds a further level of difficulty. Here we present a procedure for the production of homo- or hetero-dimeric TM peptide complexes from materials that are expressed in E. coli, thus allowing incorporation of stable isotope labels for nuclear magnetic resonance (NMR) or non-natural amino acids for other applications relatively inexpensively. The key innovation in this method is that TM complexes are produced and purified as covalently associated (disulfide-crosslinked) assemblies that can form stable, stoichiometric and homogeneous structures when reconstituted into detergent, lipid or other membrane-mimetic materials. We also present carefully optimized procedures for expression and purification that are equally applicable whether producing single TM domains or crosslinked complexes and provide advice for adapting these methods to new TM sequences.

Biophysical and biochemical studies of interactions among membrane-embedded protein domains face many technical challenges, the first of which is obtaining appropriate study material. This article describes a protocol for producing and purifying disulfide-stabilized transmembrane peptide complexes that are suitable for structural analysis by solution nuclear magnetic resonance (NMR) and other analytical applications.

Physical  interactions among the lipid-embedded alpha-helical domains of membrane  proteins play a crucial role in folding and assembly of membrane ...

Steady-state, Pre-steady-state, and Single-turnover Kinetic Measurement for DNA Glycosylase Activity

Human 8-oxoguanine DNA glycosylase (OGG1) excises the mutagenic oxidative DNA lesion 8-oxo-7,8-dihydroguanine (8-oxoG) from DNA. Kinetic characterization of OGG1 is undertaken to measure the rates of 8-oxoG excision and product release. When the OGG1 concentration is lower than substrate DNA, time courses of product formation are biphasic; a rapid exponential phase (i.e. burst) of product formation is followed by a linear steady-state phase. The initial burst of product formation corresponds to the concentration of enzyme properly engaged on the substrate, and the burst amplitude depends on the concentration of enzyme. The first-order rate constant of the burst corresponds to the intrinsic rate of 8-oxoG excision and the slower steady-state rate measures the rate of product release (product DNA dissociation rate constant, koff). Here, we describe steady-state, pre-steady-state, and single-turnover approaches to isolate and measure specific steps during OGG1 catalytic cycling. A fluorescent labeled lesion-containing oligonucleotide and purified OGG1 are used to facilitate precise kinetic measurements. Since low enzyme concentrations are used to make steady-state measurements, manual mixing of reagents and quenching of the reaction can be performed to ascertain the steady-state rate (koff). Additionally, extrapolation of the steady-state rate to a point on the ordinate at zero time indicates that a burst of product formation occurred during the first turnover (i.e. y-intercept is positive). The first-order rate constant of the exponential burst phase can be measured using a rapid mixing and quenching technique that examines the amount of product formed at short time intervals (&lt;1 sec) before the steady-state phase and corresponds to the rate of 8-oxoG excision (i.e. chemistry). The chemical step can also be measured using a single-turnover approach where catalytic cycling is prevented by saturating substrate DNA with enzyme (E&gt;S). These approaches can measure elementary rate constants that influence the efficiency of removal of a DNA lesion.

Time courses for the glycosylase activity of 8-oxoguanine DNA glycosylase are biphasic exhibiting a burst of product formation and a linear steady-state phase. Utilizing quench-flow techniques, the burst and the steady-state rates can be measured, which correspond to excision of 8-oxoguanine and release of the glycosylase from the product DNA, respectively.

Human 8-oxoguanine DNA glycosylase (OGG1) excises the mutagenic oxidative DNA lesion 8-oxo-7,8-dihydroguanine (8-oxoG) from DNA. Kinetic characterization ...

Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR

Copper (I) binding by metallochaperone transport proteins prevents copper oxidation and release of the toxic ions that may participate in harmful redox reactions. The Cu (I) complex of the peptide model of a Cu (I) binding metallochaperone protein, which includes the sequence MTCSGCSRPG (underlined is conserved), was determined in solution under inert conditions by NMR spectroscopy.
NMR is a widely accepted technique for the determination of solution structures of proteins and peptides. Due to difficulty in crystallization to provide single crystals suitable for X-ray crystallography, the NMR technique is extremely valuable, especially as it provides information on the solution state rather than the solid state. Herein we describe all steps that are required for full three-dimensional structure determinations by NMR. The protocol includes sample preparation in an NMR tube, 1D and 2D data collection and processing, peak assignment and integration, molecular mechanics calculations, and structure analysis. Importantly, the analysis was first conducted without any preset metal-ligand bonds, to assure a reliable structure determination in an unbiased manner.

Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR

The NMR-solution structure of a metallochaperone model peptide with Cu (I) was determined, and a detailed protocol from sample preparation and 1D and 2D data collection to a three-dimensional structure is described.

Copper (I) binding by metallochaperone transport proteins prevents copper oxidation and release of the toxic ions that may participate in harmful redox ...

From Constructs to Crystals &#8211; Towards Structure Determination of &#946;-barrel Outer Membrane Proteins

Membrane proteins serve important functions in cells such as nutrient transport, motility, signaling, survival and virulence, yet constitute only ~1% percent of known structures. There are two types of membrane proteins, &#945;-helical and &#946;-barrel. While &#945;-helical membrane proteins can be found in nearly all cellular membranes, &#946;-barrel membrane proteins can only be found in the outer membranes of mitochondria, chloroplasts, and Gram-negative bacteria. One common bottleneck in structural studies of membrane proteins in general is getting enough pure sample for analysis. In hopes of assisting those interested in solving the structure of their favorite &#946;-barrel outer membrane protein (OMP), general protocols are presented for the production of target &#946;-barrel OMPs at levels useful for structure determination by either X-ray crystallography and/or NMR spectroscopy. Here, we outline construct design for both native expression and for expression into inclusion bodies, purification using an affinity tag, and crystallization using detergent screening, bicelle, and lipidic cubic phase techniques. These protocols have been tested and found to work for most OMPs from Gram-negative bacteria; however, there are some targets, particularly for mitochondria and chloroplasts that may require other methods for expression and purification. As such, the methods here should be applicable for most projects that involve OMPs from Gram-negative bacteria, yet the expression levels and amount of purified sample will vary depending on the target OMP.

&#946;-barrel outer membrane proteins (OMPs) serve many functions within the outer membranes of Gram-negative bacteria, mitochondria, and chloroplasts. Here, we hope to alleviate a known bottleneck in structural studies by presenting protocols for the production of &#946;-barrel OMPs in sufficient quantities for structure determination by X-ray crystallography or NMR spectroscopy.

Membrane proteins serve important functions in cells such as nutrient transport, motility, signaling, survival and virulence, yet constitute only ~1% ...

Synthesis and Exfoliation of Discotic Zirconium Phosphates to Obtain Colloidal Liquid Crystals

Due to their abundance in natural clay and potential applications in advanced materials, discotic nanoparticles are of interest to scientists and engineers. Growth of such anisotropic nanocrystals through a simple chemical method is a challenging task. In this study, we fabricate discotic nanodisks of zirconium phosphate [Zr(HPO4)2&#183;H2O] as a model material using hydrothermal, reflux and microwave-assisted methods. Growth of crystals is controlled by duration time, temperature, and concentration of reacting species. The novelty of the adopted methods is that discotic crystals of size ranging from hundred nanometers to few micrometers can be obtained while keeping the polydispersity well within control. The layered discotic crystals are converted to monolayers by exfoliation with tetra-(n)-butyl ammonium hydroxide [(C4H9)4NOH, TBAOH]. Exfoliated disks show isotropic and nematic liquid crystal phases. Size and polydispersity of disk suspensions is highly important in deciding their phase behavior.

A two dimensional model material of discotic zirconium phosphate was developed. The inorganic crystal with lamellar structure was synthesized by hydrothermal, reflux, and microwave-assisted methods. On exfoliation with organic molecules, layered crystals can be converted to monolayers, and nematic liquid crystal phase was formed at sufficient concentration of monolayers.

Due to their abundance in natural clay and potential applications in advanced materials, discotic nanoparticles are of interest to scientists and ...

From Molecules to Materials: Engineering New Ionic Liquid Crystals Through Halogen Bonding

Herein, we demonstrate that a bottom-up approach, based on halogen bonding (XB), can be successfully applied for the design of a new type of ionic liquid crystals (ILCs). Taking advantages of the high specificity of XB for haloperfluorocarbons and the ability of anions to act as XB-acceptors, we obtained supramolecular complexes based on 1-alkyl-3-methylimidazolium iodides and iodoperfluorocarbons, overcoming the well-known immiscibility between hydrocarbons (HCs) and perfluorocarbons (PFCs). The high directionality of the XB combined with the fluorophobic effect, allowed us to obtain enantiotropic liquid crystals where a rigid, non-aromatic, XB supramolecular anion acts as mesogenic core.
X-ray structure analysis of the complex between 1-ethyl-3-methylimidazolium iodide and iodoperfluorooctane showed the presence of a layered structure, which is a manifestation of the well-known tendency to segregation of perfluoroalkyl chains. This is consistent with the observation of smectic mesophases. Moreover, all the reported complexes melt below 100 &#176;C, and most are mesomorphic even at room temperature, despite that the starting materials were non-mesomorphic in nature.
The supramolecular strategy reported here provides new design principles for mesogen design allowing a totally new class of functional materials.

A protocol for the synthesis of a new type of mesogens, based on the halogen-bonded supramolecular anion [CnF2n+1-I&#183;&#183;&#183;I&#183;&#183;&#183;I-CnF2n+1]&#8722;, is reported.

Herein, we demonstrate that a bottom-up approach, based on halogen bonding (XB), can be successfully applied for the design of a new type of ionic liquid ...

Liquid-cell Transmission Electron Microscopy for Tracking Self-assembly of Nanoparticles

Drying a nanoparticle dispersion is a versatile way to create self-assembled structures of nanoparticles, but the mechanism of this process is not fully understood. We have traced the trajectories of individual nanoparticles using liquid-cell transmission electron microscopy (TEM) to investigate the mechanism of the assembly process. Herein, we present the protocols used for liquid-cell TEM studies of the self-assembly mechanism. First, we introduce the detailed synthetic protocols used to produce uniformly sized platinum and lead selenide nanoparticles. Next, we present the microfabrication processes used to produce liquid cells with silicon nitride or silicon windows and then describe the loading and imaging procedures of the liquid-cell TEM technique. Several notes are included to provide helpful tips for the entire process, including how to manage the fragile cell windows. The individual motions of nanoparticles tracked by liquid-cell TEM revealed that changes in the solvent boundaries caused by evaporation affected the self-assembly process of nanoparticles. The solvent boundaries drove nanoparticles to primarily form amorphous aggregates, followed by flattening of the aggregates to produce a 2-dimensional (2D) self-assembled structure. These behaviors are also observed for different nanoparticle types and different liquid-cell compositions.

Here we introduce experimental protocols for the real-time observation of a self-assembly process using liquid-cell transmission electron microscopy.

Drying a nanoparticle dispersion is a versatile way to create self-assembled structures of nanoparticles, but the mechanism of this process is not fully ...

An Electrochemical Cholesteric Liquid Crystalline Device for Quick and Low-Voltage Color Modulation

We demonstrate a method for fabricating a prototype reflective display device that contains cholesteric liquid crystal (LC) as an active component. The cholesteric LC is composed of a nematic LC 4&#39;-pentyloxy-4-cyanobiphenyl (5OCB), redox-responsive chiral dopant (FcD), and a supporting electrolyte 1-ethyl-3-methylimidazolium trifluoromethanesulfonate (EMIm-OTf). The most important component is FcD. This molecule changes its helical twisting power (HTP) value in response to redox reactions. Therefore, in situ electrochemical redox reactions in the LC mixture allow for the device to change its reflection color in response to electrical stimuli. The LC mixture was introduced, by a capillary action, into a sandwich-type ITO glass cell comprising two glass slides with patterned indium tin oxide (ITO) electrodes, one of which was coated with poly(3,4-ethylenedioxythiophene)-co-poly(ethylene glycol) doped with perchlorate (PEDOT+). Upon application of +1.5 V, the reflection color of the device changed from blue (467 nm) to green (485 nm) in 0.4 s. Subsequent application of 0 V made the device recover the original blue color in 2.7 s. This device is characterized by its fastest electrical response and lowest operating voltage among any previously reported cholesteric LC device. This device could pave the way for the development of next generation reflective displays with low energy consumption rates.

A protocol for the fabrication of a reflective cholesteric liquid crystalline display device containing a redox-responsive chiral dopant allowing quick and low-voltage operation is presented.

We demonstrate a method for fabricating a prototype reflective display device that contains cholesteric liquid crystal (LC) as an active component. The ...

Preparation of Graphene-Supported Microwell Liquid Cells for In Situ Transmission Electron Microscopy

The fabrication and preparation of graphene-supported microwell liquid cells (GSMLCs) for in situ electron microscopy is presented in a stepwise protocol. The versatility of the GSMLCs is demonstrated in the context of a study about etching and growth dynamics of gold nanostructures from a HAuCl4 precursor solution. GSMLCs combine the advantages of conventional silicon- and graphene-based liquid cells by offering reproducible well depths together with facile cell manufacturing and handling of the specimen under investigation. The GSMLCs are fabricated on a single silicon substrate which drastically reduces the complexity of the manufacturing process compared to two-wafer-based liquid cell designs. Here, no bonding or alignment process steps are required. Furthermore, the enclosed liquid volume can be tailored to the respective experimental requirements by simply adjusting the thickness of a silicon nitride layer. This enables a significant reduction of window bulging in the electron microscope vacuum. Finally, a state-of-the-art quantitative evaluation of single particle tracking and dendrite formation in liquid cell experiments using only open source software is presented.

Preparation of Graphene-Supported Microwell Liquid Cells for In Situ Transmission Electron Microscopy

A protocol for preparation of graphene-supported microwell liquid cells for in situ electron microscopy of gold nanocrystals from HAuCl4 precursor solution is presented. Furthermore, an analysis routine is presented to quantify observed etching and growth dynamics.

The fabrication and preparation of graphene-supported microwell liquid cells (GSMLCs) for in situ electron microscopy is presented in a stepwise protocol. ...

High-Contrast and Fast Photorheological Switching of a Twist-Bend Nematic Liquid Crystal

Smart viscoelastic materials that respond to specific stimuli are one of the most attractive classes of materials important to future technologies, such as on-demand switchable adhesion technologies, actuators, molecular clutches, and nano-/microscopic mass transporters. Recently it was found that through a special solid-liquid transition, rheological properties can exhibit significant changes, thus providing suitable smart viscoelastic materials. However, designing materials with such a property is complex, and forward and backward switching times are usually long. Therefore, it is important to explore new working mechanisms to realize solid-liquid transitions, shorten the switching time, and enhance the contrast of rheological properties during switching. Here, a light-induced crystal-liquid phase transition is observed, which is characterized by means of polarizing light microscopy (POM), photorheometry, photo-differential scanning calorimetry (photo-DSC), and X-ray diffraction (XRD). The light-induced crystal-liquid phase transition presents key features such as (1) fast switching of crystal-liquid phases for both forward and backward reactions and (2) a high contrast ratio of viscoelasticity. In the characterization, POM is advantageous in offering information on the spatial distribution of LC molecule orientations, determining the type of liquid crystalline phases appearing in the material, and studying the orientation of LCs. Photorheometry allows measurement of a material&#8217;s rheological properties under light stimuli and can reveal the photorheological switching properties of materials. Photo-DSC is a technique to investigate thermodynamic information of materials in darkness and under light irradiation. Lastly, XRD allows studying of microscopic structures of materials. The goal of this article is to clearly present how to prepare and measure the discussed properties of a photorheological material.

This protocol demonstrates the preparation of a photorheological material that exhibits a solid phase, various liquid crystalline phases, and an isotropic liquid phase by increasing temperature. Presented here are methods for measuring the structure-viscoelasticity relationship of the material.

Smart viscoelastic materials that respond to specific stimuli are one of the most attractive classes of materials important to future technologies, such ...