This work was supported by grant-in-aid for Young Scientists (16K17956) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, The Kyoto Technoscience Center, and The Mitani Foundation for Research and Development.

1. Instruments
<ol>
	<li>Polarization device
	<ol>
		<li>To construct a polarization device, provide a light source with a halogen lamp, a light guide, polarizers, a sample stage, an optical rail, rod stands, and a digital single-lens reflex camera (see Figure 1C and Materials List for the polarization device parts).</li>
	</ol>
	</li>
</ol>
2. Preparation of the Biopolymer Solution
<ol>
	<li>Polysaccharide solutions
	<ol>
		<li>Dissolve sacran7 (0.5 g) in pure water (100 mL) by stirring at ~80 &#176;C for more than 12 h. During the dissolution, cover the container with plastic wrap to prevent evaporation. Prepare an aqueous solution of xanthan gum in the same manner.</li>
		<li>Cool the solutions at ~25 &#176;C to obtain 0.5 wt% aqueous solutions.</li>
		<li>Centrifuge the sacran solution to remove impurities (48,400 x g, 4 &#176;C, 1 h, 3 times).</li>
	</ol>
	</li>
	<li>MT solution
	<ol>
		<li>Prepare a 0.5 wt% tubulin solution (1 mL) in a Britton-Robinson buffer (80 mM piperazine-N,N&#8242;-bis(2-ethanesulfonic acid) (PIPES); 1 mM ethylene glycol-bis(&#946;-aminoethyl ether)-N,N,N&#39;,N&#39;-tetraacetic acid (EGTA); and 5 mM MgCl2, pH 6.8) on ice8.</li>
		<li>Use 0.5 wt% tubulin solution (50 &#181;L) and guanosine-5&#39;-[(&#945;,&#946;)-methyleno]triphosphate (GpCpp) (5 &#181;L) to prepare a GpCpp-containing tubulin solution (50 &#181;L). Incubate at 37 &#176;C for 3 h to obtain a stable MT nucleus. 
		NOTE: The role of GpCpp is to support MT formation and to completely suppress the depolymerization of MT to tubulin.</li>
		<li>Mix the 0.5 wt% tubulin solution (950 &#181;L) and the GpCpp-containing tubulin solution (50 &#181;L) at ~25 &#176;C for 1 day to obtain a stable 0.5 wt% MT solution.</li>
	</ol>
	</li>
	<li>DNA solution
	<ol>
		<li>Prepare a 0.5 wt% DNA solution (1 mL) in Tris-EDTA buffer solution (10 mM Tris, pH 8.0, with 1 mM EDTA).</li>
	</ol>
	</li>
	<li>Keep the 0.5 wt% biopolymer sample solutions at 25 &#176;C for the drying experiment.</li>
</ol>
3. Drying Experiments and Observation under Cross-polarized Light
<ol>
	<li>Solutions in a one-side-open cell (Figure 1A)
	<ol>
		<li>Cut a silicon sheet (see Materials List) into an appropriate shape with a thickness of 1 mm (inner dimension of 5-15 mm, 1 mm, and ~20 mm; Figure 1A).
		<ol>
			<li>Assemble a one-side-open cell composed of a silicon spacer with inner dimension of 5-15 mm, 1 mm, and ~20 mm and two non-modified glass slides (76 mm &#215; 1 mm &#215; 26 mm). Fix both sides of the cell with double clips in advance to keep the sample solution from leaking out.</li>
		</ol>
		</li>
		<li>Slowly add each of the 0.5 wt% biopolymer solution (100-300 &#181;L) using a ~1-mm pore-size pipette tip to each cell at ~25 &#176;C. Remove air bubbles from the cells using a syringe needle.</li>
		<li>Place the cells in an oven with an air circulator at 60 &#176;C under atmospheric pressure for evaporation; the evaporation direction is opposite to that of gravity.</li>
	</ol>
	</li>
	<li>Observations under cross-polarized light (Figure 1B-1C)
	<ol>
		<li>Provide straight visible light via a 100 W halogen lamp with a flat-surface light source over a wide area (80 mm x 80 mm). Adjust the polarizers to 45&#176; and 135&#176; using the holders (Figure 1C).</li>
		<li>Fix the positions of the light source, polarizers, sample stage, and camera using an optical rail and rod stands (Figure 1C). Place the sample stage between the two polarizers (the distance between the polarizers should be ~5 cm). Place the camera ~20 cm from the sample stage to allow focusing.</li>
		<li>At given times, place the samples from step 3.1.3 between the polarizers on the stage parallel to the XZ-plane and cover the device with a black curtain; the actual device is shown in Figure 1C.</li>
		<li>Photograph the samples through linear crossed polarizers using a digital single-lens reflex camera with a standard zoom lens (see Materials List). Control the camera settings, such as focal distance, using computer software (see Materials List).</li>
	</ol>
	</li>
	<li>Spatio-temporal analysis of the transmitted light intensity (Figure 1C)
	<ol>
		<li>To evaluate the change of the orientational order parameter&#160;in the drying process, collect photographs hourly for 24 h.</li>
		<li>Measure the transmitted light intensity along the centerline in the Z-direction as a gray value using an image processing program (e.g., ImageJ).</li>
		<li>Plot a graph of the gray value as a function of the distance from the upper open side.</li>
	</ol>
	</li>
	<li>Microscopic observations under cross-polarized light (Figure 1D)
	<ol>
		<li>To check the methods, make microscopic observations with a polarization microscope equipped with a CCD camera9. Keep a first-order retardation plate in the light path. Control the conditions for the photos using a PC software.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Flemming, H. -. C., Wingender, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biofilm+matrix.">The biofilm matrix.</a> Nat. Rev. Microbiology. 8 (9), 623-633 (2010).</li><li>Osada, Y., Kawamura, R., Sano, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Hydrogels of cytoskeletal proteins. , (2016).</li><li>Rothemund, P. W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Folding+DNA+to+create+nanoscale+shapes+and+patterns.">Folding DNA to create nanoscale shapes and patterns.</a> Nature. 440, 297-302 (2006).</li><li>Okeyoshi, K., Okajima, M. K., Kaneko, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Milliscale+self-integration+of+megamolecule+biopolymers+on+a+drying+gas-aqueous+liquid+crystalline+interface.">Milliscale self-integration of megamolecule biopolymers on a drying gas-aqueous liquid crystalline interface.</a> Biomacromolecules. 17 (6), 2096-2103 (2016).</li><li>De Gennes, P. G., Prost, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The physics of liquid crystals. , (1993).</li><li>De Gennes, P. G., Brochard-Wyart, F., Quere, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Capillarity and wetting phenomena: drops, bubbles, pearls, waves. , (2003).</li><li>Okajima, M. K., Kaneko, D., Mitsumata, T., Kaneko, T., Watanabe, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyanobacteria+that+produce+megamolecules+with+efficient+self-orientations.">Cyanobacteria that produce megamolecules with efficient self-orientations.</a> Macromolecules. 42 (8), 3058-3062 (2009).</li><li>Okeyoshi, K., Kawamura, R., Yoshida, R., Osada, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermo-+and+photo-enhanced+microtubule+formation+from+Ru(bpy)32+-conjugated+tubulin.">Thermo- and photo-enhanced microtubule formation from Ru(bpy)32+-conjugated tubulin.</a> J. Mater. Chem. B. 2, 41-45 (2014).</li><li>Matsuo, E. S., Tanaka, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetics+of+discontinuous+volume-phase+transition+of+gels.">Kinetics of discontinuous volume-phase transition of gels.</a> J. Chem. Phys. 89, 1695-1703 (1988).</li><li>Okajima, K., Mishima, R., Amornwachirabodee, K., Mitsumata, T., Okeyoshi, K., Kaneko, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anisotropic+swelling+in+hydrogels+formed+by+cooperatively+aligned+megamolecules.">Anisotropic swelling in hydrogels formed by cooperatively aligned megamolecules.</a> RSC Adv. 5, 86723-86729 (2015).</li><li>Joshi, G., Okeyoshi, K., Okajima, M. K., Kaneko, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directional+control+of+diffusion+and+swelling+in+megamolecular+polysaccharide+hydrogels.">Directional control of diffusion and swelling in megamolecular polysaccharide hydrogels.</a> Soft Matter. 12, 5515-5518 (2016).</li></ol>

The authors have nothing to disclose.

It was sometimes difficult for the camera to focus on the sample due to the transmitted light intensity being too low. In such cases, placing an extended transparent plastic film on the stage helped to arrange the focus. The limitation of the observable resolution was dependent on the camera lens, ~10 &#181;m in this case. The observable limitation of the sample thickness, &#916;y, was dependent on the maximum light intensity of the lamp, ~10 mm in this case.
The advantage of the device shown ...

By focusing on the rigid, rod-shaped structures of biopolymers, dynamic soft materials have been used for various applications, including polysaccharide biofilm matrices1, &#34;active gels&#34; composed of cytoskeletal proteins2, and &#34;DNA origami&#34; of desired shapes3. To clarify the structural properties, many strategies have been explored, such as transmission electron microscopy, scanning electron microscopy, atomic force microscopy, and confocal fluorescence microscopy. However, because these methods are mostly undertaken in a dried or static state, it is difficult to explain the dynamic behaviors in macroscopic scales, as seen in actual living systems. Recently, we successfully observed the dynamic behavior of biopolymers on the aqueous air-LC interface through polarized light4. During the visualization of the oriented structure while drying the biopolymer solution, the temporal changes indicated self-integration of biopolymers on the unstable air-LC interface.
Here, we describe a protocol for the drying of LC biopolymer solutions at the air-LC interface using polarized instruments. As opposed to other analyses of the LC phase that do not consider drying5,6, the LC dynamics during the drying process were investigated here by evaluating the orientational order parameter&#160;&#160;in the lateral view of the fluid phase in a one-side-open cell. The combination of the cell evaporation and the use of polarized instruments allowed for macroscopic monitoring with a controlled evaporation direction. In addition, it was possible to validate the drying records by focusing on the crystalline structures of the adsorbed microdomains, which were affected by molecular weight, concentration, etc. To demonstrate the effectiveness of the method, the drying processes of basic biopolymers with rigid rod shapes, such as polysaccharides, microtubules (MTs), and DNA, were investigated. We chose these biopolymers because they are typical examples of hierarchical macromolecules with megamolecular weights, and their intermolecular interactions enable them to form LC states.

<table border="0" cellpadding="0" cellspacing="0" width="617">
	<tbody>
		<tr height="69">
			<td height="69" style="height:69px;width:172px;">sacran</td>
			<td style="width:151px;">Green Science Materials Inc., Japan</td>
			<td style="width:131px;"></td>
			<td style="width:163px;">From Aphanothece sacrum. 
			Mw = 1.9 &#215; 107 g mol-1</td>
		</tr>
		<tr height="69">
			<td height="69" style="height:69px;width:172px;">xanthan gum</td>
			<td style="width:151px;">Taiyo Kagaku Co., Japan</td>
			<td style="width:131px;">Neosoft XC</td>
			<td style="width:163px;">From Xanthomonas campestris. 
			Mw = 4.7 &#215; 106 g mol-1</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:172px;">tubulin</td>
			<td style="width:151px;">Cytoskeleton, Inc., USA</td>
			<td style="width:131px;">T240</td>
			<td style="width:163px;">From porcine brain.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:172px;">GpCpp</td>
			<td style="width:151px;">Jena Bioscience, Germany</td>
			<td style="width:131px;">NU405L</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:172px;">piperazine-N,N&#8242;-bis(2-ethanesulfonic acid)</td>
			<td style="width:151px;">Sigma-Aldrichi, Co. LLC.</td>
			<td style="width:131px;">P6757-500G</td>
			<td style="width:163px;">PIPES</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:172px;">ethylene glycol-bis(&#946;-aminoethyl ether)-N,N,N&#39;,N&#39;-tetraacetic acid</td>
			<td style="width:151px;">Dojindo Molecular Technologies, Inc.</td>
			<td style="width:131px;">342-01314</td>
			<td style="width:163px;">EGTA</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:172px;">MgCl2-6H2O</td>
			<td style="width:151px;">Wako Pure Chemical Industries, Ltd.</td>
			<td style="width:131px;">135-15055</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:172px;">KOH</td>
			<td style="width:151px;">Wako Pure Chemical Industries, Ltd.</td>
			<td style="width:131px;">162-21813</td>
			<td style="width:163px;">pellet</td>
		</tr>
		<tr height="21">
			<td height="58" style="height:58px;width:172px;">DNA</td>
			<td rowspan="2" style="width:151px;">Sigma-Aldrich, Co. LLC.</td>
			<td rowspan="2" style="width:131px;">D1626</td>
			<td rowspan="2" style="width:163px;">From salmon testes. 
			Mw = 1.3 &#215; 106 Da (~2000 bp)</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:172px;">Tris-EDTA buffer solution</td>
			<td style="width:151px;">Sigma-Aldrich, Co. LLC.</td>
			<td style="width:131px;">T9285-100ML</td>
			<td style="width:163px;">10 mM Tris, pH 8.0, with 1 mM EDTA</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:172px;">slide glass</td>
			<td style="width:151px;">Matsunami Glass Ind., Ltd., Japan</td>
			<td style="width:131px;">S1111</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:172px;">silicon rubber sheet</td>
			<td style="width:151px;">Asone Co.</td>
			<td style="width:131px;">6-611-32</td>
			<td style="width:163px;">Thickness: 1 mm</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:172px;">centrifuge</td>
			<td style="width:151px;">Beckman-Coulter, Inc., USA</td>
			<td style="width:131px;">Avanti J-25</td>
			<td style="width:163px;">equipped with a JA-20 rotor</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:172px;">light source</td>
			<td style="width:151px;">Sumita Optical Glass, Inc., Japan</td>
			<td style="width:131px;">LS-LHA</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:172px;">light guide</td>
			<td style="width:151px;">Sumita Optical Glass, Inc., Japan</td>
			<td style="width:131px;">GF7.2-1-L1500R-M80 (AAAR-015M)</td>
			<td style="width:163px;">80 mm &#215; 80 mm</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:172px;">halogen lamp</td>
			<td style="width:151px;">Ushio Inc., Japan</td>
			<td style="width:131px;">JCR 15V150WBN</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:172px;">polarizer</td>
			<td style="width:151px;">Luceo, Co.,Ltd.</td>
			<td style="width:131px;">POLAX-42S</td>
			<td style="width:163px;">40 x 80 x 2t (45&#730;)</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:172px;">holder</td>
			<td style="width:151px;">Sigmakoki, Co.,Ltd.</td>
			<td style="width:131px;">KMH-80</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:172px;">sample stage</td>
			<td style="width:151px;">Sigmakoki, Co.,Ltd.</td>
			<td style="width:131px;">TARW-25503L</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:172px;">sample holder</td>
			<td style="width:151px;">Sigmakoki, Co.,Ltd.</td>
			<td style="width:131px;">SHA-25RO</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:172px;">rod</td>
			<td style="width:151px;">Sigmakoki, Co.,Ltd.</td>
			<td style="width:131px;">ROU-12-40</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:172px;">posts holder</td>
			<td style="width:151px;">Sigmakoki, Co.,Ltd.</td>
			<td style="width:131px;">RS-6-40</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:172px;">posts holder</td>
			<td style="width:151px;">Sigmakoki, Co.,Ltd.</td>
			<td style="width:131px;">RS-12-60</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:172px;">posts holder</td>
			<td style="width:151px;">Sigmakoki, Co.,Ltd.</td>
			<td style="width:131px;">RS-12-80</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:172px;">posts holder</td>
			<td style="width:151px;">Sigmakoki, Co.,Ltd.</td>
			<td style="width:131px;">RS-12-130</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:172px;">carrier</td>
			<td style="width:151px;">Sigmakoki, Co.,Ltd.</td>
			<td style="width:131px;">CAA-25LS</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:172px;">camera holder</td>
			<td style="width:151px;">Sigmakoki, Co.,Ltd.</td>
			<td style="width:131px;">CMH-2</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:172px;">medium optical rail</td>
			<td style="width:151px;">Sigmakoki, Co.,Ltd.</td>
			<td style="width:131px;">OBA-500SH</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:172px;">lenstube</td>
			<td style="width:151px;">Tomytech, BORG</td>
			<td style="width:131px;">lenstube BK</td>
			<td style="width:163px;">80&#966;, L25 mm&#160;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:172px;">lenstube</td>
			<td style="width:151px;">Tomytech, BORG</td>
			<td style="width:131px;">lenstube BK</td>
			<td style="width:163px;">80&#966;, L50 mm&#160;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:172px;">multiband</td>
			<td style="width:151px;">Tomytech, BORG</td>
			<td style="width:131px;"></td>
			<td style="width:163px;">80&#966;&#160;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:172px;">V plate</td>
			<td style="width:151px;">Tomytech, BORG</td>
			<td style="width:131px;">V plate 60S</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:172px;">plate holder</td>
			<td style="width:151px;">Viexen, Co.,Ltd.</td>
			<td style="width:131px;">plate holder SX</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:172px;">EOS Kiss X7i&#160;</td>
			<td style="width:151px;">Canon Inc., Japan</td>
			<td style="width:131px;">8594B001</td>
			<td style="width:163px;">with a standard zoom lens,&#160; EFP 18-55 mm</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:172px;">photographic software</td>
			<td style="width:151px;">Canon Inc., Japan</td>
			<td style="width:131px;">EOS Utility</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:172px;">PC</td>
			<td style="width:151px;">Microsoft</td>
			<td style="width:131px;">Surface</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:172px;">polarization microscope</td>
			<td style="width:151px;">Olympus</td>
			<td style="width:131px;">BX51</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:172px;">first order retardation plate</td>
			<td style="width:151px;">Olympus</td>
			<td style="width:131px;">U-TP530</td>
			<td style="width:163px;">&#955; = 530 nm</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:172px;">CCD camera</td>
			<td style="width:151px;">Olympus</td>
			<td style="width:131px;">DP80</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:172px;">photographic software</td>
			<td style="width:151px;">Olympus</td>
			<td style="width:131px;">cellSens Standard</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:172px;">Java-based image processing program</td>
			<td style="width:151px;">the National Institutes of Health</td>
			<td style="width:131px;">ImageJ</td>
			<td style="width:163px;"></td>
		</tr>
	</tbody>
</table>

methods for the self-integration of megamolecular biopolymers on the drying air-lc interface

Living organisms that use water are always prone to drying in the environment. Their activities are driven by biopolymer-based micro- and macro-structures, as seen in the cases of moving water in vascular bundles and moisturizing water in skin layers. In this study, we developed a method for assessing the effect of aqueous liquid crystalline (LC) solutions composed of biopolymers on drying. As LC biopolymers have megamolecular weight, we chose to study polysaccharides, cytoskeletal proteins, and DNA. The observation of biopolymer solutions during drying under polarized light reveals milliscale self-integration starting from the unstable air-LC interface. The dynamics of the aqueous LC biopolymer solutions can be monitored by evaporating water from a one-side-open cell. By analyzing the images taken using cross-polarized light, it is possible to recognize the spatio-temporal changes in the orientational order parameter. This method can be useful for the characterization of not only artificial materials in various fields, but also natural living tissues. We believe that it will provide an evaluation method for soft materials in the biomedical and environmental fields.

Using the device as shown in Figure 1, the self-integration from microdomain to macrodomain on a drying air-LC interface was evaluated (Figure 2A). As the first demonstration of the drying experiment, two kinds of megamolecular polysaccharides, sacran (Mw = 1.9 x 107 g mol-1) and xanthan gum (4.7 x 106 g mol-1), were compared. Figure 2B shows photographs of the solutions in t...

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Methods for the Self-integration of Megamolecular Biopolymers on the Drying Air-LC Interface

A method for the drying-induced self-integration of megamolecular biopolymers on the air-liquid crystalline interface is provided here. This methodology will be useful not only for understanding the macroscopic potentials of biopolymers, but also as an evaluation method for soft materials in biomedical and environmental fields.

Living organisms that use water are always prone to drying in the environment. Their activities are driven by biopolymer-based micro- and ...

methods-for-self-integration-megamolecular-biopolymers-on-drying-air

Research

JoVE Journal

Bioengineering

6.0K Views.  Japan Advanced Institute of Science and Technology. A method for the drying-induced self-integration of megamolecular biopolymers on the air-liquid crystalline interface is provided here. This methodology will be useful not only for understanding the macroscopic potentials of biopolymers, but also as an evaluation method for soft materials in biomedical and environmental fields.

Video: Methods for the Self-integration of Megamolecular Biopolymers on the Drying Air-LC Interface

Bridging the Bio-Electronic Interface with Biofabrication

Advancements in lab-on-a-chip technology promise to revolutionize both research and medicine through lower costs, better sensitivity, portability, and higher throughput. The incorporation of biological components onto biological microelectromechanical systems (bioMEMS) has shown great potential for achieving these goals. Microfabricated electronic chips allow for micrometer-scale features as well as an electrical connection for sensing and actuation. Functional biological components give the system the capacity for specific detection of analytes, enzymatic functions, and whole-cell capabilities. Standard microfabrication processes and bio-analytical techniques have been successfully utilized for decades in the computer and biological industries, respectively. Their combination and interfacing in a lab-on-a-chip environment, however, brings forth new challenges. There is a call for techniques that can build an interface between the electrode and biological component that is mild and is easy to fabricate and pattern.
Biofabrication, described here, is one such approach that has shown great promise for its easy-to-assemble incorporation of biological components with versatility in the on-chip functions that are enabled. Biofabrication uses biological materials and biological mechanisms (self-assembly, enzymatic assembly) for bottom-up hierarchical assembly. While our labs have demonstrated these concepts in many formats 1,2,3, here we demonstrate the assembly process based on electrodeposition followed by multiple applications of signal-based interactions. The assembly process consists of the electrodeposition of biocompatible stimuli-responsive polymer films on electrodes and their subsequent functionalization with biological components such as DNA, enzymes, or live cells 4,5. Electrodeposition takes advantage of the pH gradient created at the surface of a biased electrode from the electrolysis of water 6,7,. Chitosan and alginate are stimuli-responsive biological polymers that can be triggered to self-assemble into hydrogel films in response to imposed electrical signals 8. The thickness of these hydrogels is determined by the extent to which the pH gradient extends from the electrode. This can be modified using varying current densities and deposition times 6,7. This protocol will describe how chitosan films are deposited and functionalized by covalently attaching biological components to the abundant primary amine groups present on the film through either enzymatic or electrochemical methods 9,10. Alginate films and their entrapment of live cells will also be addressed 11. Finally, the utility of biofabrication is demonstrated through examples of signal-based interaction, including chemical-to-electrical, cell-to-cell, and also enzyme-to-cell signal transmission. 
Both the electrodeposition and functionalization can be performed under near-physiological conditions without the need for reagents and thus spare labile biological components from harsh conditions. Additionally, both chitosan and alginate have long been used for biologically-relevant purposes 12,13. Overall, biofabrication, a rapid technique that can be simply performed on a benchtop, can be used for creating micron scale patterns of functional biological components on electrodes and can be used for a variety of lab-on-a-chip applications.

This article describes a biofabrication approach: deposition of stimuli-responsive polysaccharides in the presence of biased electrodes to create biocompatible films which can be functionalized with cells or proteins. We demonstrate a bench-top strategy for the generation of the films as well as their basic uses for creating interactive biofunctionalized surfaces for lab-on-a-chip applications.

Advancements in lab-on-a-chip technology promise to revolutionize both research and medicine through lower costs, better sensitivity, portability, and ...

Flexural Rigidity Measurements of Biopolymers Using Gliding Assays

Microtubules are cytoskeletal polymers which play a role in cell division, cell mechanics, and intracellular transport. Each of these functions requires microtubules that are stiff and straight enough to span a significant fraction of the cell diameter. As a result, the microtubule persistence length, a measure of stiffness, has been actively studied for the past two decades1. Nonetheless, open questions remain: short microtubules are 10-50 times less stiff than long microtubules2-4, and even long microtubules have measured persistence lengths which vary by an order of magnitude5-9.
	Here, we present a method to measure microtubule persistence length. The method is based on a kinesin-driven microtubule gliding assay10. By combining sparse fluorescent labeling of individual microtubules with single particle tracking of individual fluorophores attached to the microtubule, the gliding trajectories of single microtubules are tracked with nanometer-level precision. The persistence length of the trajectories is the same as the persistence length of the microtubule under the conditions used11. An automated tracking routine is used to create microtubule trajectories from fluorophores attached to individual microtubules, and the persistence length of this trajectory is calculated using routines written in IDL.
	This technique is rapidly implementable, and capable of measuring the persistence length of 100 microtubules in one day of experimentation. The method can be extended to measure persistence length under a variety of conditions, including persistence length as a function of length along microtubules. Moreover, the analysis routines used can be extended to myosin-based acting gliding assays, to measure the persistence length of actin filaments as well.

A method to measure the persistence length or flexural rigidity of biopolymers is described. The method uses a kinesin-driven microtubule gliding assay to experimentally determine the persistence length of individual microtubules and is adaptable to actin-based gliding assays.

Microtubules are cytoskeletal polymers which  play a role in cell division, cell mechanics, and intracellular transport. Each of these functions requires ...

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain central aspects of cellular physiology. To easily combine cell patterning with Multi-Electrode Arrays (MEAs) and silicon-based &#8216;lab on a chip&#8217; technologies, a microfabrication-compatible protocol is required. We describe a method that utilizes deposition of the polymer parylene-C on SiO2&#160;wafers. Photolithography enables accurate and reliable patterning of parylene-C at micron-level resolution. Subsequent activation by immersion in fetal bovine serum (or another specific activation solution) results in a substrate in which cultured cells adhere to, or are repulsed by, parylene or SiO2 regions respectively. This technique has allowed patterning of a broad range of cell types (including primary murine hippocampal cells, HEK 293 cell line, human neuron-like teratocarcinoma cell line, primary murine cerebellar granule cells, and primary human glioma-derived stem-like cells). Interestingly, however, the platform is not universal; reflecting the importance of cell-specific adhesion molecules. This cell patterning process is cost effective, reliable, and importantly can be incorporated into standard microfabrication (chip manufacturing) protocols, paving the way for integration of microelectronic technology.

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

This protocol describes a microfabrication-compatible method for cell patterning on SiO2. A predefined parylene-C design is photolithographically printed on SiO2 wafers. Following incubation with serum (or other activation solution) cells adhere specifically to (and grow according to the conformity of) underlying parylene-C, whilst being repulsed by SiO2 regions.

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain ...

Molecular Entanglement and Electrospinnability of Biopolymers

Electrospinning is a fascinating technique to fabricate micro- to nano-scale fibers from a wide variety of materials. For biopolymers, molecular entanglement of the constituent polymers in the spinning dope was found to be an essential prerequisite for successful electrospinning. Rheology is a powerful tool to probe the molecular conformation and interaction of biopolymers. In this report, we demonstrate the protocol for utilizing rheology to evaluate the electrospinnability of two biopolymers, starch and pullulan, from their dimethyl sulfoxide (DMSO)/water dispersions. Well-formed starch and pullulan fibers with average diameters in the submicron to micron range were obtained. Electrospinnability was evaluated by visual and microscopic observation of the fibers formed. By correlating the rheological properties of the dispersions to their electrospinnability, we demonstrate that molecular conformation, molecular entanglement, and shear viscosity all affect electrospinning. Rheology is not only useful in solvent system selection and process optimization, but also in understanding the mechanism of fiber formation on a molecular level.

Electrospinning is a fascinating technique used to fabricate micro- to nano-scale fibers from a wide variety of materials. Molecular entanglement of the constituent polymers in the spinning dope is essential for successful electrospinning. We present a protocol for utilizing rheology to evaluate the electrospinnability of two biopolymers, starch and pullulan.

Electrospinning is a fascinating technique to fabricate micro- to nano-scale fibers from a wide variety of materials. For biopolymers, molecular ...

Gene Transfection toward Spheroid Cells on Micropatterned Culture Plates for Genetically-modified Cell Transplantation

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. The cell spheroids are prepared in a culture system on micropatterned plates coated with a thermosensitive polymer. A number of spheroids are formed on the plates, corresponding to the cell adhesion areas of 100 &#181;m diameter that are regularly arrayed in a two-dimensional manner, surrounded by non-adhesive areas that are coated by a polyethylene glycol (PEG) matrix. The spheroids can be easily recovered as a liquid suspension by lowering the temperature of the plates, and their structure is well maintained by passing them through injection needles with a sufficiently large caliber (over 27 G). Genetic modification is achieved by gene transfection using the original non-viral gene carrier, polyplex nanomicelle, which is capable of introducing genes into cells without disrupting the spheroid structure. For primary hepatocyte spheroids transfected with a luciferase-expressing gene, the luciferase is sustainably obtained in transplanted animals, along with preserved hepatocyte function, as indicated by albumin expression. This system can be applied to a variety of cell types including mesenchymal stem cells.

This protocol describes a cell transplantation system using genetically modified, injectable spheroids. Cell spheroids are cultured on micropatterned culture plates and recovered after gene introduction using polyplex nanomicelles. This system facilitates prolonged transgene expression from the transplanted cells in host animals while maintaining the innate function of the cells.

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. ...

Methods for Characterizing the Co-development of Biofilm and Habitat Heterogeneity

Biofilms are surface-attached microbial communities that have complex structures and produce significant spatial heterogeneities. Biofilm development is strongly regulated by the surrounding flow and nutritional environment. Biofilm growth also increases the heterogeneity of the local microenvironment by generating complex flow fields and solute transport patterns. To investigate the development of heterogeneity in biofilms and interactions between biofilms and their local micro-habitat, we grew mono-species biofilms of Pseudomonas aeruginosa and dual-species biofilms of P. aeruginosa and Escherichia coli under nutritional gradients in a microfluidic flow cell. We provide detailed protocols for creating nutrient gradients within the flow cell and for growing and visualizing biofilm development under these conditions. We also present protocols for a series of optical methods to quantify spatial patterns in biofilm structure, flow distributions over biofilms, and mass transport around and within biofilm colonies. These methods support comprehensive investigations of the co-development of biofilm and habitat heterogeneity.

Biofilms have complex interactions with their surrounding environment. To comprehensively investigate biofilm-environment interactions, we present here a series of methods to create heterogeneous chemical environment for biofilm development, to quantify local flow velocity, and to analyze mass transport in and around biofilm colonies.

Biofilms are surface-attached microbial communities that have complex structures and produce significant spatial heterogeneities. Biofilm development is ...

PIP-on-a-chip: A Label-free Study of Protein-phosphoinositide Interactions

Numerous cellular proteins interact with membrane surfaces to affect essential cellular processes. These interactions can be directed towards a specific lipid component within a membrane, as in the case of phosphoinositides (PIPs), to ensure specific subcellular localization and/or activation. PIPs and cellular PIP-binding domains have been studied extensively to better understand their role in cellular physiology. We applied a pH modulation assay on supported lipid bilayers (SLBs) as a tool to study protein-PIP interactions. In these studies, pH sensitive ortho-Sulforhodamine B conjugated phosphatidylethanolamine is used to detect protein-PIP interactions. Upon binding of a protein to a PIP-containing membrane surface, the interfacial potential is modulated (i.e. change in local pH), shifting the protonation state of the probe. A case study of the successful usage of the pH modulation assay is presented by using phospholipase C delta1 Pleckstrin Homology (PLC-&#948;1 PH) domain and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) interaction as an example. The apparent dissociation constant (Kd,app) for this interaction was 0.39 &#177; 0.05 &#181;M, similar to Kd,app values obtained by others. As previously observed, the PLC-&#948;1 PH domain is PI(4,5)P2 specific, shows weaker binding towards phosphatidylinositol 4-phosphate, and no binding to pure phosphatidylcholine SLBs. The PIP-on-a-chip assay is advantageous over traditional PIP-binding assays, including but not limited to low sample volume and no ligand/receptor labeling requirements, the ability to test high- and low-affinity membrane interactions with both small and large molecules, and improved signal to noise ratio. Accordingly, the usage of the PIP-on-a-chip approach will facilitate the elucidation of mechanisms of a wide range of membrane interactions. Furthermore, this method could potentially be used in identifying therapeutics that modulate protein&#39;s capacity to interact with membranes.

Here we present a supported lipid bilayer in the context of a microfluidic platform to study protein-phosphoinositide interactions using a label-free method based on pH modulation.

Numerous cellular proteins interact with membrane surfaces to affect essential cellular processes. These interactions can be directed towards a specific ...

Microhoneycomb Monoliths Prepared by the Unidirectional Freeze-drying of Cellulose Nanofiber Based Sols: Method and Extensions

Monolithic honeycomb structures have been attractive to multidisciplinary fields due to their high strength-to-weight ratio. Particularly, microhoneycomb monoliths (MHMs) with micrometer-scale channels are expected as efficient platforms for reactions and separations because of their large surface areas. Up to now, MHMs have been prepared by a unidirectional freeze-drying (UDF) method only from very limited precursors. Herein, we report a protocol from which a series of MHMs consisting of different components can be obtained. Recently, we found that cellulose nanofibers function as a distinct structure-directing agent towards the formation of MHMs through the UDF process. By mixing the cellulose nanofibers with water soluble substances which do not yield MHMs, a variety of composite MHMs can be prepared. This significantly enriches the chemical constitution of MHMs towards versatile applications.

Here, we present a general protocol to prepare a variety of microhoneycomb monoliths (MHMs) in which fluid can pass through with an extremely low pressure drop. MHMs obtained are expected to be used as filters, catalyst supports, flow-type electrodes, sensors and scaffolds for biomaterials.

Monolithic honeycomb structures have been attractive to multidisciplinary fields due to their high strength-to-weight ratio. Particularly, microhoneycomb ...

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 ...

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

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

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