The authors thank D. Glass, H. Kim, N. Cira, A. Choksi, S. Rajan, and B. Keys for their helpful suggestions and the Spormann lab for access to their confocal microscope. Furthermore, the authors acknowledge the support from Stanford Bio-X Bowes and NSERC PGS fellowships, the National Institute of Health (R21-AI-139941), and the American Cancer Society (RSG-14-177-01).

1. Preparation of pDawn-Ag43 Bacterial Strains
<ol>
	<li>Transform pDawn-Ag43 into an E. coli strain of interest (Figure 1).
	<ol>
		<li>Grow a cloning strain hosting pDawn-Ag43 plasmid (obtainable from a plasmid repository) by inoculating the strain in LB broth supplemented with 50 &#956;g/mL spectinomycin (LB+spec) in a culture tube (overnight in a shaking incubator at ~250 rpm, 37 &#176;C). Then, use a miniprep kit to harvest the purified pDawn-Ag43 plasmid10.</li>
		<li>Choose an E. coli strain of interest to be patterned. Thus far, pDawn-Ag43 has been verified to work in MG16553 and BW25113.</li>
		<li>Use an established protocol to generate competent (e.g., chemically competent11 or electrocompetent12) stocks of the chosen E. coli strain (e.g., MG1655).</li>
		<li>Transform pDawn-Ag43 plasmid into the competent bacteria11,12, followed by 1 h recovery, and plate&#160;on LB+spec agar plates. Allow colonies to grow overnight at 37 &#176;C.</li>
	</ol>
	</li>
	<li>Store the pDawn-Ag43 transformed strains.
	<ol>
		<li>Inoculate a single colony of pDawn-Ag43 from an LB+spec agar plate into LB+spec broth in a culture tube and grow it in a shaking incubator (at ~250 rpm, 37 &#176;C) to exponential phase (OD600 ~0.4 - 0.8).</li>
		<li>Prepare stock to store the pDawn-Ag43 transformed strain at -80 &#176;C long-term by mixing 1 mL of culture with 1 mL of 50% sterile glycerol in a cryo-tube to obtain a 25% glycerol freezer stock. Store this in a -80 &#176;C freezer.</li>
		<li>If additional plasmids (e.g., fluorescent reporter plasmids) need to be transformed, create competent cells11,12 from the pDawn-Ag43 transformed strain and repeat the transformation process11,12 for additional plasmids as needed. 
		NOTE: If using electroporation8, it is possible to cotransform multiple plasmids (including pDawn-Ag43) simultaneously; however, a simultaneous transformation is not recommended using chemical transformation methods, as the large size (&#62; 10 kB) of pDawn-Ag43 means transformation efficiencies are reduced.</li>
	</ol>
	</li>
</ol>
2. Preparation of the Projector Optical Set-up for Illuminating Bacteria
<ol>
	<li>Obtain a bacterial incubator with non-transparent walls and a hole for passing cables, a portable projector capable of fitting and functioning inside the bacterial incubator, and a laptop equipped with software for presentation-projection (see Table of Materials). When choosing the projector and incubator, ensure that the minimal focus distance of the projector is less than the interior height of the incubator.</li>
	<li>Place the projector inside the incubator at the bottom, with the aperture pointing directly upward, at the ceiling, where the biofilm culture chamber is attached (Figure 2).</li>
	<li>Fix the projector in place by constructing a set-up with an optical breadboard base connected to a vertical post, in turn connected to a horizontal post which screws into the projector (Figure 2). Note that this set-up can be modified depending on the projector/available parts. Ultimately, the key requirement is that the projector is solidly fixed near the bottom of the incubator, with the aperture pointing upward.</li>
	<li>Connect the laptop via the display cable (e.g., HDMI) to the projector inside the incubator.</li>
	<li>If the surfaces&#8212;especially the ceiling&#8212;of the interior of the incubator are reflective (e.g., metal polished surface), cover them with dark matte surfaces to minimize reflections.</li>
	<li>Use tape to attach an empty &#39;dummy&#39; culture dish to the ceiling of the incubator. Note that, as with the projector, there are multiple acceptable ways to attach a culture dish; ensure that the transparent bottom surface of the culture chamber, where illumination occurs, is not covered.</li>
	<li>Adjust the focus on the projector by turning the focusing knob so that the focusing plane coincides with the bottom surface of the biofilm culture dish (e.g., a well plate) attached to the ceiling of the incubator (Figure 2). The projector should illuminate a sharp, non-blurry image onto the bottom of the culture dish. Remove the &#39;dummy&#39; culture dish once the projection has been optimized.</li>
	<li>Using laptop software, direct the projector to illuminate the full field of view with maximum blue illumination (e.g., RGB = [0, 0, 255]) by presenting a full blue slide.</li>
	<li>Measure the illumination intensity of the projector using an optical power meter by placing the photodetector head at the incubator ceiling and reading the intensity on the corresponding power meter calibrated to light wavelength 460 nm. Follow the instructions on connecting and calibrating the photodetector for the specific power meter used.
	<ol>
		<li>Reduce ambient light (e.g., turn off room lights, or place the incubator away from light sources) as much as possible prior to making illumination intensity measurements.</li>
	</ol>
	</li>
	<li>Adjust the illumination intensity using an adjustable neutral density filter placed at the projector aperture. Rotate the filter to adjust the illumination intensity measured by the power meter, until the illumination intensity in the center of the blue-light projected region reads 50 &#956;W/cm2. 
	NOTE: While it is possible to adjust the illumination intensity by using lower blue RGB values on the software end, using a filter while maximizing the blue RGB value has the advantage of maximizing the optical contrast of the system between illuminated vs. dark regions.</li>
	<li>Draw arbitrary patterns using the presentation/projector software on the laptop and display these patterns on the ceiling of the incubator, using the projector. 
	NOTE: Biofilm-forming regions should be drawn with maximum blue illumination (e.g., RGB = [0, 0, 255]), non-biofilm-forming regions with no illumination (e.g., RGB = [0, 0, 0]).</li>
</ol>
3. Culturing Patterned Biofilms
<ol>
	<li>Prior to illumination, prepare the pDawn-Ag43 bacteria, starting from the glycerol freezer stock, so that they are reliably induced at the late exponential growth phase for illumination (Figure 3A).
	<ol>
		<li>Streak a pDawn-Ag43 strain from glycerol stock onto LB+spec agar plates. Allow colonies to grow overnight (37 &#176;C). 
		NOTE: From here until the illumination culture step, ensure the cells stay in the dark as much as possible&#8212;brief periods at ambient illumination (e.g., for subdilution) are acceptable.</li>
		<li>Inoculate a colony of pDawn-Ag43 bacteria from an agar plate in LB+spec broth and grow the culture overnight to&#160;stationary phase (in a shaking incubator at ~250 rpm, 37 &#176;C, for ~16 h).</li>
		<li>Subdilute the culture at a ratio of 1:1,000 with LB+spec broth (e.g.,&#160;add 1 &#956;L of overnight culture to 1 mL of fresh LB+spec).</li>
		<li>Allow the subdiluted culture to grow until&#160;late exponential/early stationary phase (OD ~1.0, ~6 h in a shaking incubator at ~250 rpm, 37 &#176;C).</li>
		<li>While waiting for the culture to grow, prepare M63 media with 1x M63 salts, 1 mM MgSO4, 0.2% glucose, 0.1% casamino acids, and 50 &#956;g/mL spectinomycin in water (ensure the constituent parts are sterile).</li>
		<li>Subdilute the late-exponential-phase culture at a ratio of 1:100 with M63 media supplemented with 50 &#956;g/mL spectinomycin. Then, introduce the dilution into a biofilm culture dish (e.g., pipette the diluted sample into a well plate). 
		NOTE: The volumes required for this 1:100 subdilution depend on the culture dish being used (e.g., if using a 6-well polystyrene well plate [non-tissue-culture treated], a single sample would require adding 20 &#956;L of culture to 2 mL of M63+spec, as the standard well volume of a 6-well plate is ~2 mL).</li>
	</ol>
	</li>
	<li>As the samples are now ready for illumination, tape the culture dish to the ceiling of the incubator, ensuring that the surface at the bottom of the dish is transparent for illumination from below by the projector. 
	NOTE: It is important to ensure that the ceiling of the incubator is not reflective, to minimize stray illumination. Stray illumination can also be reduced by using black-walled plates as culture dishes, although this is not strictly necessary&#8212;if using such plates, ensure the bottom surface is transparent.</li>
	<li>Allow the biofilms to culture in the incubator overnight (16 h with no shaking, at 37 &#176;C). Note that some projectors become less reliable at higher temperatures. If that is the case, culture at lower temperatures (e.g., 30 &#176;C), keeping in mind that the incubation time may need to be&#160;increased, depending on the E. coli strain.</li>
</ol>
4. Imaging Patterned Biofilms
<ol>
	<li>After the overnight growth of the biofilm samples, remove the culture dish from the incubator. The dish will have biofilm bacteria attached to its bottom surface where it has been illuminated, as well as planktonic bacteria dispersed in the liquid media above.</li>
	<li>Discard the planktonic cells by removing the liquid media from the culture dish (e.g., by gently aspirating with a pipette).</li>
	<li>Rinse the sample 2x with a phosphate-buffered saline (PBS) solution to remove the remaining planktonic cells (Figure 3B) by gently pipetting in PBS, followed by aspiration.</li>
	<li>If the cells are fluorescently tagged, directly image the samples using fluorescence microscopy13 (e.g., wide-field13, 3-D confocal14, etc.). 
	NOTE: Fluorescent biofilms can also be preserved using a self-hardening mounting medium. Apply one drop of mounting media to a biofilm sample, cover it with a glass coverslip, taking care not to capture any bubbles underneath, and allow it to harden overnight before imaging.</li>
	<li>If the bacterial cells used are not fluorescently tagged, apply the crystal violet stain technique15 (Figure 3B) to enhance biofilm contrast prior to imaging.</li>
</ol>
5. Protocol Modifications/Alternatives
<ol>
	<li>Grow pDawn-Ag43 bacteria on different surfaces.
	<ol>
		<li>Put glass or poly-dimethyl-siloxane (PDMS) coupons (e.g., coverslips or thin strips of PDMS) into well plates prior to the addition of a bacterial sample/illumination, and follow the same protocol as before to pattern pDawn-ag43 biofilms on glass and PDMS.</li>
	</ol>
	</li>
	<li>Grow pDawn-Ag43 bacteria inside a transparent, enclosed culture chamber.
	<ol>
		<li>Generate a culture chamber mold for PDMS.
		<ol>
			<li>For a basic culture chamber, generate mold by attaching a hard, rectangular prism to a flat surface (the prism will become a cavity in the PDMS that serves as the culture chamber once the mold is cast). Note that more intricate culture chamber molds can be fabricated using soft lithography16.</li>
		</ol>
		</li>
		<li>Cast a PDMS cavity by pouring PDMS into the mold and allowing it to cure (for a detailed soft lithography protocol, see JoVE&#39;s Science Education Database16).</li>
		<li>After curing, trim any excess PDMS, punch inlet/outlet channels into the cavity/culture chamber and bond the cavity to a flat surface (e.g., glass/polystyrene) by firmly pressing the PDMS onto the flat surface, leaving the cavity between the surface and the PDMS ceiling as the biofilm culture chamber. 
		NOTE: More permanent bonding based on plasma treatment16 can also be used, but the chips will then not be reusable.</li>
		<li>Follow the culture protocol as before, using a syringe with blunt tip needles (instead of a pipette) to introduce the bacterial sample into the culture chamber/rinse with PBS buffer (Figure 3C).
		<ol>
			<li>If using a temporarily bonded cavity, use only negative pressure to pull liquid into/out of the chamber, to ensure that the cavity does not become unbonded from the underlying glass/polystyrene surface.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Grow pDawn-Ag43 bacteria using a film photomask for structured illumination.
	<ol>
		<li>Design a biofilm pattern using CAD software compatible with a film photomask printer/print service. The photomask film design should be clear in regions where the biofilm is meant to be printed, and black/opaque elsewhere. When complete, send the photomask file to the printer/printing service and await the return of the physical photomask.</li>
		<li>Instruct the projector to illuminate a full field of view with maximum blue illumination (e.g., RGB = [0, 0, 255]) using laptop software.</li>
		<li>Cut out a region of interest from the larger film photomask, and tape it directly to the bottom of the biofilm culture dish prior to introducing the bacterial sample for overnight illumination (Figure 3D). Culture the biofilms as before and remove the photomask after culturing, prior to imaging.</li>
	</ol>
	</li>
</ol>

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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilm+Lithography+enables+high-resolution+cell+patterning+via+optogenetic+adhesin+expression.">Biofilm Lithography enables high-resolution cell patterning via optogenetic adhesin expression.</a> Proceedings of the National Academy of Sciences of the United States of America. 115 (14), 3698-3703 (2018).</li><li>Ohlendorf, R., Vidavski, R. 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The authors have nothing to disclose.

In light of the need for research tools that allow for biofilm structure control, we have presented an easy-to-use protocol for patterning bacterial biofilms using the pDawn-Ag43 optogenetic construct. With this technique, E. coli biofilms can be optically patterned on various surface environments, including enclosed chambers, with a spatial resolution below 25 &#956;m.
Overall, this protocol can be broken down into four main sections: (1) the preparation of the pDawn-Ag43 bacteria, (2) the preparation of the optical and culture set-up hardware, (3) the pre-illumination bacterial growth steps, and (4) the post-illumination rinses and imaging.
The critical part of section 1 is the successful transformation of pDawn-Ag43 plasmid into the E. coli strain of interest. This is facilitated by isolating high-quality purified plasmid and generating high-quality competent cells for transformation (Table 1, troubleshooting).
The critical part of section 2 is the optimization of the projector set-up so that the illumination intensity is adjusted to 50 &#956;W/cm2 at the 460-nm wavelength, and the projector is properly focused at the biofilm sample height. Note that in this protocol, we describe an inverted illumination set-up where the projector shines light from below, upward toward the biofilm sample. The advantage of this set-up is that the light only needs to travel through the bottom of the culture dish before reaching the biofilm formation surface. Illumination from above means that the light would have to travel through the liquid media above the biofilm surface, which, during the course of the growth, gets cloudy with planktonic cells. In addition to these concerns, it is also important to minimize stray light in the optical set-up as much as possible, for example, by covering up reflective surfaces on the interior of the incubator&#8212;this helps to obtain sharper patterned biofilms. On a related note, sharper biofilm patterns can also be obtained by using a photomask to control illumination patterning (Figure 3D, Figure 4C). Common issues requiring troubleshooting include projector reliability issues at higher temperatures (e.g., 37 &#176;C), which can be minimized by incubating the biofilm growth at lower temperatures (e.g., 30 &#176;C), as well as computer software that causes operating system updates or blue light filtering during overnight growth (Table 1). It is also important to note that, depending on the projector and incubator model used, it is also possible that heat generated from the projector will result in a higher interior temperature than the incubator set temperature, which may need to be corrected.
The critical part of section 3 is obtaining reliable and repeatable bacterial samples before they are induced by illumination. For this reason, it is recommended to obtain clonal colonies of pDawn-Ag43 bacteria by streaking them out on an agar plate and then using the liquid culture steps to ensure that the bacteria are illuminated/induced at the late exponential growth phase in a repeatable manner.
Finally, the critical part of section 4 is to thoroughly, but also gently, wash away the planktonic cells remaining after the biofilm patterning protocol; thus, it is recommended to perform multiple gentle rinse steps with PBS.
Compared to existing techniques for cell patterning5,6, optical biofilm patterning based on pDawn-Ag43 has a reasonably low barrier of entry to use, in that it does not require microfabrication, clean-room facilities, complex chemistry, or surface pretreatment, yet is still able to pattern with the high resolution (25 &#956;m) typically associated with microfabrication techniques. The method extends previous work on bacterial photolithography for controlling gene expression17. Currently, pDawn-Ag43 plasmid is limited to E. coli, as it uses a pUC-based origin of replication, but pDawn and Ag43 are both compatible in other (Gram-negative) bacterial species. Genetic techniques are available for potentially introducing light-regulated biofilm formation to different bacterial species and represents a possible direction for future&#160;research. Another potential limitation of the technique is that it works by increasing biofilm formation in strains with weak native biofilm formation (e.g., MG1655 E. coli). However, strains with strong native biofilm formation have biofilms form regardless of illumination conditions, precluding patterned biofilm formation using pDawn-Ag43 as described here; yet optogenetic techniques may still prove applicable in regulating biofilm formation. We note that in other contexts, alternative methods of biofilm patterning may be available, such as via optical c-di-GMP modulation18.
Overall, pDawn-Ag43 based patterning will be appropriate for use in applications that investigate the effect of biofilm structure on function1 and, therefore, could benefit from tunable control over biofilm patterning&#8212;a particularly relevant example to highlight is the study of microbial ecology in biofilms2. Future directions include making patterned biomaterials8,9 and/or structured bacterial communities. Alternative applications of this accessible protocol also include bio-art19, given the clear aesthetic potential, as well as formal and informal life science education20,21,22. From an educational perspective, the protocol described here combines many relevant techniques (bacterial culture, transformation, optics/optogenetics) and is also modularly extendable (e.g., include microfluidics).

Biofilms are surface-attached communities of microbes, and are well-known for their strong structure-function coupling. Spatial geometry and patterning of biofilms play an important role in overall community function (and vice versa)1. The small length scales involved in biofilm structure&#8212;on the order of tens of microns2&#8212;make tunable and convenient control of biofilm patterning a challenging problem. Here we demonstrate a protocol that allows for biofilms to be precisely patterned in arbitrary geometries, based on optical illumination.
The protocol presented here uses pDawn-Ag433, an optogenetic construct that couples biofilm formation in E. coli bacteria to optical illumination by driving the expression of Ag43 (an adhesin gene responsible for surface adhesion and biofilm formation) under the control of pDawn4 (a transcriptional regulator controlled by optical illumination). The method is convenient to use and can pattern biofilms on various surface environments, including enclosed (transparent) culture chambers. Compared to existing cell deposition methods, such as droplet-based deposition5 or surface prepatterning/treatment6, pDawn-Ag43 does not require microfabrication or clean-room facilities and does not require materials beyond those available to a typical microbiology laboratory. It is able to pattern with a spatial resolution below 25 &#956;m, approaching the spatial dimensions of microcolonies in naturally existing biofilms2. Overall, this technique provides the ability to manipulate biofilm structure, which then opens many avenues to study microecology in bacterial communities7. Additionally, patterned biofilms may provide a convenient platform upon which to engineer useful biomaterials8,9. In this paper, we discuss the basic protocol required for patterning biofilms using pDawn-Ag43 and address potential modifications and troubleshooting related to the method.

<table>
	<tbody>
		<tr>
			<td>DH5alpha/pDawn-Ag43</td>
			<td>Addgene</td>
			<td>107742</td>
			<td>DH5alpha cloning strain hosting pDawn-Ag43 plasmid - plasmid needs to be moved to E. coli strain of interest prior to use</td>
		</tr>
		<tr>
			<td>MG1655 E. coli</td>
			<td>Coli Genetic Stock Center - Yale University</td>
			<td>CGSC #6300</td>
			<td>MG1655 was used as E. coli strain of interest in this paper&#39;s representative results</td>
		</tr>
		<tr>
			<td>RFP expression plasmid</td>
			<td>iGEM biobricks</td>
			<td>J04450-pSB4K5bb</td>
			<td>Many options exist to obtain fluorescent bacteria - if using plasmid, ensure backbone does not conflict with colE1 ori of pDawn-Ag43</td>
		</tr>
		<tr>
			<td>Plasmid miniprep kit</td>
			<td>Qiagen</td>
			<td>27104</td>
			<td></td>
		</tr>
		<tr>
			<td>LB broth powder</td>
			<td>Affymetrix</td>
			<td>75852</td>
			<td>Add 20 g/L to water, autoclave, add 50 &#956;g/mL spectinomycin to get sterile LB+spec</td>
		</tr>
		<tr>
			<td>LB agar powder</td>
			<td>Affymetrix</td>
			<td>75851</td>
			<td>Add 35 g/L to water, autoclave, add 50 &#956;g/mL spectinomycin, pour into petri dishes to get sterile LB+spec plates</td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>Fisherbrand</td>
			<td>431760</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectinomycin hydrochloride pentahydrate</td>
			<td>abcam</td>
			<td>ab141968</td>
			<td>Make 1000x stock 50mg/mL in water, filter sterilize and dilute into media as needed</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G5516</td>
			<td>Mix at 1:1 ratio with water, sterilize by autoclave or filter to obtain 50% glycerol</td>
		</tr>
		<tr>
			<td>M63 media salts 5X solution</td>
			<td>Bio-world</td>
			<td>705729</td>
			<td>Add cas-amino acids, glucose and MgSO4, bring to 1X salts concentration by adding sterile water</td>
		</tr>
		<tr>
			<td>Casamino acids</td>
			<td>Amresco</td>
			<td>J851</td>
			<td>Make 20% stock in water, filter sterilize and add to M63 as supplement (final concentration 0.1%)</td>
		</tr>
		<tr>
			<td>D-glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G8270</td>
			<td>Make 20% stock in water, filter sterilize and add to M63 as supplement (final concentration 0.2%)</td>
		</tr>
		<tr>
			<td>Magnesium sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>M7506</td>
			<td>Make 1 M stock in water, autoclave and add to M63 as supplement (final concentration 1 mM)</td>
		</tr>
		<tr>
			<td>Crystal violet&#160;</td>
			<td>Acros organics</td>
			<td>212120250</td>
			<td>Dilute to 0.1% in water prior to use</td>
		</tr>
		<tr>
			<td>Self-hardening mounting media (Shandon immumount)</td>
			<td>Thermo Scientific</td>
			<td>9990402</td>
			<td>Use to preserve samples over long term for fluorescence imaging</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS) solution&#160;</td>
			<td>Gibco</td>
			<td>10010023</td>
			<td>Can also use PBS prepared from powder / tablets</td>
		</tr>
		<tr>
			<td>6 well plate</td>
			<td>Fisherbrand</td>
			<td>351146</td>
			<td>Used as biofilm culture dish for representative results</td>
		</tr>
		<tr>
			<td>PDMS kit</td>
			<td>Dow</td>
			<td>SYLGARD 184</td>
			<td>Can be used to make enclosed microchamber cavities using soft lithography</td>
		</tr>
		<tr>
			<td>1 mL syringe</td>
			<td>BD syringe</td>
			<td>309659</td>
			<td>For use with liquid handling with enclosed microchambers</td>
		</tr>
		<tr>
			<td>Blunt tip needle</td>
			<td>CML supply</td>
			<td>901-23-050</td>
			<td>Attaches to 1 mL syringe</td>
		</tr>
		<tr>
			<td>Lab tape</td>
			<td>Fisherbrand</td>
			<td>15-901</td>
			<td>Use to attach culture chamber to incubator ceiling</td>
		</tr>
		<tr>
			<td>Bacterial incubator</td>
			<td>Sheldon Manufacturing</td>
			<td>SMI6</td>
			<td>Ensure interior height of incubator is tall enough to focus projector at the ceiling</td>
		</tr>
		<tr>
			<td>Portable projector</td>
			<td>Ivation</td>
			<td>IV-PJ-PRO-4-1</td>
			<td>Many portable projector models exist, pDawn-Ag43 has been tested with multiple models including LED/laser based, with blue light channel ranging from 450-460nm central wavelength</td>
		</tr>
		<tr>
			<td>Optical breadboard base</td>
			<td>ThorLabs</td>
			<td>MSB6</td>
			<td>Base for optical setup to hold projector - many other setups possible, just need to hold projector firmly at bottom of incubator, pointing upwards</td>
		</tr>
		<tr>
			<td>Optical post</td>
			<td>ThorLabs</td>
			<td>TR8</td>
			<td>2 posts needed - one to be set up vertically extending out of breadboard base, one horizontally attached via right-angle clamp</td>
		</tr>
		<tr>
			<td>Optical post right-angle clamp</td>
			<td>ThorLabs</td>
			<td>RA90</td>
			<td>Connects vertical and horizontal posts</td>
		</tr>
		<tr>
			<td>Mounting base</td>
			<td>ThorLabs</td>
			<td>BA1S</td>
			<td>Connects optical breadboard base and vertical post</td>
		</tr>
		<tr>
			<td>1/4&#34;-20 screw</td>
			<td>ThorLabs</td>
			<td>SH25S050</td>
			<td>Attaches vertical post to mounting base, mounting base to breadboard base</td>
		</tr>
		<tr>
			<td>1/4&#34;-20 set-screw</td>
			<td>ThorLabs</td>
			<td>SS25E63D</td>
			<td>Connects horizontal post to projector via tripod screw-hole</td>
		</tr>
		<tr>
			<td>Optical power meter</td>
			<td>Newport</td>
			<td>840</td>
			<td>Use with power meter detector to measure projector illumination intensity - many power meter models exist, using one that has extendable detector will facilitate measurement</td>
		</tr>
		<tr>
			<td>Optical power meter detector</td>
			<td>Newport</td>
			<td>818-UV</td>
			<td>Connects to power meter (above) - UV detector not strictly necessary as blue light is within visible range</td>
		</tr>
		<tr>
			<td>Adjustable ND filter</td>
			<td>K&#38;F Concept</td>
			<td>SKU0689</td>
			<td>Adjustable (by rotating) neutral density filter - place above projector aperture</td>
		</tr>
		<tr>
			<td>Presentation-projector software</td>
			<td>Microsoft</td>
			<td>Powerpoint</td>
			<td>Any software that allows drawing / presentation will suffice</td>
		</tr>
		<tr>
			<td>CAD software</td>
			<td>Autodesk&#160;</td>
			<td>AutoCAD</td>
			<td>Used for designing photomasks, many mask printing services are compatible with AutoCAD files</td>
		</tr>
		<tr>
			<td>Film photomask</td>
			<td>Fineline Imaging</td>
			<td>n/a</td>
			<td>Many photomask printer services exist for high resolution (&#62;30000DPI) film photomask printing</td>
		</tr>
	</tbody>
</table>

high-resolution patterned biofilm deposition using pdawn-ag43

Spatial structure and patterning play an important role in bacterial biofilms. Here we demonstrate an accessible method for culturing E. coli biofilms into arbitrary spatial patterns at high spatial resolution. The technique uses a genetically encoded optogenetic construct&#8212;pDawn-Ag43&#8212;that couples biofilm formation in E. coli to optical stimulation by blue light. We detail the process for transforming E. coli with&#160;pDawn-Ag43, preparing the required optical set-up, and the protocol for culturing patterned biofilms using pDawn-Ag43 bacteria. Using this protocol, biofilms with a spatial resolution below 25 &#956;m can be patterned on various surfaces and environments, including enclosed chambers, without requiring microfabrication, clean-room facilities, or surface pretreatment. The technique is convenient and appropriate for use in applications that investigate the effect of biofilm structure, providing tunable control over biofilm patterning. More broadly, it also has potential applications in biomaterials, education, and bio-art.

As seen in Figure 4A, pDawn-Ag43 bacteria were used to generate biofilms patterned in polystyrene well plates with projector illumination (the projector was set to illuminate a polka-dot pattern), imaged through brightfield microscopy with crystal violet stain as a contrast agent, and fluorescence microscopy using red-fluorescent-protein-expressing bacteria. Fluorescent biofilm samples can also be imaged using confocal microscopy14 to obtain images of the biofilm in 3-D (Figure 4B). In Figure 4C, we illustrate the high-resolution patterning possible by using a film photomask to provide patterned illumination to the biofilm sample. Finally, in Figure 4D and 4E, we demonstrate examples of patterning on glass and PDMS surfaces, as well as enclosed PDMS culture chambers&#8212;these illustrate the different types of environments where pDawn-Ag43 patterning can be applied.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58625/58625fig1.jpg" /> 
Figure 1: Preparation of pDawn-Ag43 bacteria (protocol section 1). Preparing pDawn-Ag43 bacteria capable of light-regulated biofilm formation involves purifying pDawn-Ag43 plasmid from a host cloning strain, transforming it into an E. coli strain of interest, and creating bacterial freezer stock for long-term storage. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/58625/58625fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58625/58625fig2.jpg" /> 
Figure 2: Preparation of an optical set-up for biofilm sample illumination (protocol section 2). The optical set-up is housed inside a bacterial incubator and consists of a computer-connected projector illuminating a biofilm sample. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/58625/58625fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58625/58625fig3.jpg" /> 
Figure 3: Culture protocol for patterning biofilms (protocol section 3). (A) Prior to illumination, pDawn-Ag43 bacteria are prepared prior to patterning such that they are reliably induced at the proper growth phase. (B) After overnight illuminated growth, a patterned biofilm will be present at the bottom of the culture dish, along with planktonic cells in the liquid media, and after some further processing, the biofilm is ready for imaging. (C) As an alternative to well plates, biofilms can be cultured in enclosed culture chambers such as a molded PDMS cavity. In this case, syringes attached to blunt tip needles can be used to introduce the sample and flush liquids out of the chamber. (D) As an alternative to projector-based illumination patterns, patterns can also be generated by taping film photomasks directly to the bottom of biofilm culture chambers. In this case, the projector should be set up to illuminate blue light across the full field. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/58625/58625fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58625/58625fig4.jpg" /> 
Figure 4: Representative results of biofilms patterned using pDawn-Ag43. All results were obtained using an MG1655 E. coli host strain. (A) pDawn-Ag43 bacteria were used to generate biofilms patterned in polystyrene well plates with projector illumination (the projector was set to illuminate a polka-dot pattern), imaged through brightfield microscopy with crystal violet stain as a contrast agent, and fluorescence microscopy using red-fluorescent-protein-expressing bacteria. (B) Fluorescent biofilm samples are imaged with confocal microscopy to obtain 3-D images of the biofilm. (C) High-resolution biofilms can be patterned with a film photomask to provide patterned illumination to the biofilm sample. (D) Biofilms can be patterned on glass and PDMS surfaces. (E) Biofilms can be patterned in enclosed culture chambers. This figure has been adapted from previous work3. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/58625/58625fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Problem</td>
			<td>Potential causes/solutions</td>
		</tr>
		<tr>
			<td>Tranforming pDawn-Ag43 into host strain - no colonies</td>
			<td>Low plasmid concentration - check plasmid concentration on spectrometer. A typical miniprep of pDawn-Ag43 should yield at least 100 ng/&#956;L; use up to 10-100 ng for transformation</td>
		</tr>
		<tr>
			<td>Check/remake competent cells: competent cells should have transformation efficiency at least 10^6 cfu/&#181;g verified using a standard plasmid such as pUC19 - if not, remake competent cells</td>
		</tr>
		<tr>
			<td>Wrong (level of) antibiotic on LB agar plate - make sure to use 50 &#956;g/mL spectinomycin&#160; for selection</td>
		</tr>
		<tr>
			<td>Projector illumination turns off / inconsistent overnight</td>
			<td>Disable problematic software such as: automatic overnight software/OS updating, night-time blue-light filter</td>
		</tr>
		<tr>
			<td>Projector may be overheating - set incubator to lower temperature while projector is turned on (e.g. 30 &#176;C instead of 37 &#176;C) - note projector as heat source can overheat incubator beyond set point</td>
		</tr>
		<tr>
			<td>Remove unnecessary sources of humidity from incubator, as these may affect projector electronics</td>
		</tr>
		<tr>
			<td>No/low levels of biofilm formed after overnight illumination, no&#160; planktonic cells growth either (i.e. liquid is clear)</td>
			<td>Wrong (level of) antibiotic&#160; - make sure to use 50 &#956;g/mL spectinomycin</td>
		</tr>
		<tr>
			<td>Check everything is added to M63 recipe properly&#160;</td>
		</tr>
		<tr>
			<td>No/low levels of biofilm formed after overnight illumination, only planktonic cells (i.e. liquid is cloudy)</td>
			<td>Check light level, projector should be illuminating blue light at 50 &#956;W/cm^2 at 460 nm wavelength&#160;</td>
		</tr>
		<tr>
			<td>Try letting bacteria grow for shorter/longer time after 1:1000 LB subdilution step prior to adding to M63</td>
		</tr>
		<tr>
			<td>Restreak bacteria on LB plate, start from fresh colony to generate overnight stationary phase culture</td>
		</tr>
		<tr>
			<td>Ensure projector is working consistently overnight - see point above</td>
		</tr>
		<tr>
			<td>Fuzzy biofilm patterns, high levels of background noise</td>
			<td>Reduce stray light from optical illumination system, cover reflective surfaces on interior of incubator</td>
		</tr>
		<tr>
			<td>Consider using photomask-based (as opposed to projector-based) structured illumination&#160;</td>
		</tr>
		<tr>
			<td>Check projector is properly focused at the bottom surface of the biofilm culture chamber</td>
		</tr>
	</tbody>
</table>
Table 1: Common troubleshooting issues.

Watch this Scientific Journal Video about High-resolution Patterned Biofilm Deposition Using pDawn-Ag43 at JoVE.com

High-resolution Patterned Biofilm Deposition Using pDawn-Ag43

We demonstrate a method for depositing Escherichia coli bacterial biofilms in arbitrary spatial patterns with a high resolution using optical stimulation of a genetically encoded surface-adhesion construct.

Spatial structure and patterning play an important role in bacterial biofilms. Here we demonstrate an accessible method for culturing E. coli biofilms ...

high-resolution-patterned-biofilm-deposition-using-pdawn-ag43

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

9.3K Views.  Stanford University. We demonstrate a method for depositing Escherichia coli bacterial biofilms in arbitrary spatial patterns with a high resolution using optical stimulation of a genetically encoded surface-adhesion construct.

Video: High-resolution Patterned Biofilm Deposition Using pDawn-Ag43

Patterned Photostimulation with Digital Micromirror Devices to Investigate Dendritic Integration Across Branch Points

Light is a versatile and precise means to control neuronal excitability. The recent introduction of light sensitive effectors such as channel-rhodopsin and caged neurotransmitters have led to interests in developing better means to control patterns of light in space and time that are useful for experimental neuroscience. One conventional strategy, employed in confocal and 2-photon microscopy, is to focus light to a diffraction limited spot and then scan that single spot sequentially over the region of interest. This approach becomes problematic if large areas have to be stimulated within a brief time window, a problem more applicable to photostimulation than for imaging. An alternate strategy is to project the complete spatial pattern on the target with the aid of a digital micromirror device (DMD). The DMD approach is appealing because the hardware components are relatively inexpensive and is supported by commercial interests. Because such a system is not available for upright microscopes, we will discuss the critical issues in the construction and operations of such a DMD system. Even though we will be primarily describing the construction of the system for UV photolysis, the modifications for building the much simpler visible light system for optogenetic experiments will also be provided. The UV photolysis system was used to carryout experiments to study a fundamental question in neuroscience, how are spatially distributed inputs integrated across distal dendritic branch points. The results suggest that integration can be non-linear across branch points and the supralinearity is largely mediated by NMDA receptors.

Digital micromirror devices (DMD) can generate complex patterns in time and space with which to control neuronal excitability. Issues relevant to the design, construction, and operation of DMD systems are discussed. Such a system enabled the demonstration of non-linear integration across distal dendritic branch points.

Light is a versatile and precise means to control neuronal excitability. The recent introduction of light sensitive effectors such as channel-rhodopsin ...

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution. Traditionally, multiple experiments are run in parallel, with individual samples removed from the study at sequential time points for evaluation by light microscopy. Several intravital techniques have been developed, with confocal, multiphoton, and second harmonic microscopy all demonstrating their ability to be used for imaging in situ 1. With these systems, however, the required infrastructure is complex and expensive, involving scanning laser systems and complex light sources. Here we present a protocol for the design and assembly of a high-resolution microendoscope which can be built in a day using off-the-shelf components for under US$5,000. The platform offers flexibility in terms of image resolution, field-of-view, and operating wavelength, and we describe how these parameters can be easily modified to meet the specific needs of the end user.
We and others have explored the use of the high-resolution microendoscope (HRME) in in vitro cell culture 2-5, in excised 6 and living animal tissues 2,5, and in human tissues in vivo 2,7. Users have reported the use of several different fluorescent contrast agents, including proflavine 2-4, benzoporphyrin-derivative monoacid ring A (BPD-MA) 5, and fluoroscein 6,7, all of which have received full, or investigational approval from the FDA for use in human subjects. High-resolution microendoscopy, in the form described here, may appeal to a wide range of researchers working in the basic and clinical sciences. The technique offers an effective and economical approach which complements traditional benchtop microscopy, by enabling the user to perform high-resolution, longitudinal imaging in situ.

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

In many biological and clinical situations it is advantageous to study cellular processes as they evolve in their native microenvironment. Here we describe the assembly and use of a low-cost fiber-optic microscope which can provide real time imaging in cell culture, animal studies, and clinical patient studies.

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution.  ...

High Resolution 3D Imaging of Ex-Vivo Biological Samples by Micro CT

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized tomography (&mu;CT).
High resolution &mu;CT is a very versatile yet accurate (1-2 microns of resolution) technique for 3D examination of ex-vivo biological samples1, 2. As opposed to electron tomography, the &mu;CT allows the examination of up to 4 cm thick samples. This technique requires only few hours of measurement as compared to weeks in histology. In addition, &mu;CT does not rely on 2D stereologic models, thus it may complement and in some cases can even replace histological methods3, 4, which are both time consuming and destructive. Sample conditioning and positioning in &mu;CT is straightforward and does not require high vacuum or low temperatures, which may adversely affect the structure. The sample is positioned and rotated 180&deg; or 360&deg;between a microfocused x-ray source and a detector, which includes a scintillator and an accurate CCD camera, For each angle a 2D image is taken, and then the entire volume is reconstructed using one of the different available algorithms5-7. The 3D resolution increases with the decrease of the rotation step. The present video protocol shows the main steps in preparation, immobilization and positioning of the sample followed by imaging at high resolution.

High Resolution 3D Imaging of Ex-Vivo Biological Samples by Micro CT

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized tomography ( CT).

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized ...

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Continuous High-resolution Microscopic Observation of Replicative Aging in Budding Yeast

We demonstrate the use of a simple microfluidic setup, in which single budding yeast cells can be tracked throughout their entire lifespan. The microfluidic chip exploits the size difference between mother and daughter cells using an array of micropads. Upon loading, cells are trapped underneath these micropads, because the distance between the micropad and cover glass is similar to the diameter of a yeast cell (3-4 &mu;m). After the loading procedure, culture medium is continuously flushed through the chip, which not only creates a constant and defined environment throughout the entire experiment, but also flushes out the emerging daughter cells, which are not retained underneath the pads due to their smaller size. The setup retains mother cells so efficiently that in a single experiment up to 50 individual cells can be monitored in a fully automated manner for 5 days or, if necessary, longer. In addition, the excellent optical properties of the chip allow high-resolution imaging of cells during the entire aging process.

We describe here the operation of a microfluidic device that allows continuous and high-resolution microscopic imaging of single budding yeast cells during their complete replicative and/or chronological lifespan.

We demonstrate the use of a simple microfluidic setup, in which single budding yeast cells can be tracked throughout their entire lifespan. The ...

High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., kinetics, coexistence of different states and populations, transient interactions), which are typically hidden in ensemble measurements, such as those obtained with standard biochemical or microscopy methods. Thus, dynamic events, such as receptor-receptor interactions, can be followed in real time in a living cell with high spatiotemporal resolution. This protocol describes a method based on labeling with small and bright organic fluorophores and total internal reflection fluorescence (TIRF) microscopy to directly visualize single receptors on the surface of living cells. This approach allows one to precisely localize receptors, measure the size of receptor complexes, and capture dynamic events such as transient receptor-receptor interactions. The protocol provides a detailed description of how to perform a single-molecule experiment, including sample preparation, image acquisition and image analysis. As an example, the application of this method to analyze two G-protein-coupled receptors, i.e., &#946;2-adrenergic and &#947;-aminobutyric acid type B (GABAB) receptor, is reported. The protocol can be adapted to other membrane proteins and different cell models, transfection methods and labeling strategies.

This protocol describes how to use total internal reflection fluorescence microscopy to visualize and track single receptors on the surface of living cells and thereby analyze receptor lateral mobility, size of receptor complexes as well as to visualize transient receptor-receptor interactions. This protocol can be extended to other membrane proteins.

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., ...

Preparation of Mica Supported Lipid Bilayers for High Resolution Optical Microscopy Imaging

Supported lipid bilayers (SLBs) are widely used as a model for studying membrane properties (phase separation, clustering, dynamics) and its interaction with other compounds, such as drugs or peptides. However SLB characteristics differ depending on the support used. 
Commonly used techniques for SLB imaging and measurements are single molecule fluorescence microscopy, FCS and atomic force microscopy (AFM). Because most optical imaging studies are carried out on a glass support, while AFM requires an extremely flat surface (generally mica), results from these techniques cannot be compared directly, since the charge and smoothness properties of these materials strongly influence diffusion. Unfortunately, the high level of manual dexterity required for the cutting and gluing thin slices of mica to the glass slide presents a hurdle to routine use of mica for SLB preparation. Although this would be the method of choice, such prepared mica surfaces often end up being uneven (wavy) and difficult to image, especially with small working distance, high numerical aperture lenses. Here we present a simple and reproducible method for preparing thin, flat mica surfaces for lipid vesicle deposition and SLB preparation. Additionally, our custom made chamber requires only very small volumes of vesicles for SLB formation. The overall procedure results in the efficient, simple and inexpensive production of high quality lipid bilayer surfaces that are directly comparable to those used in AFM studies.

We present a method of preparing mica supported lipid bilayers for high resolution microscopy. Mica is transparent and flat on an atomic scale, but rarely used in imaging because of handling difficulties; our preparation results in even deposition of the mica sheet, and reduces the material used in bilayer preparation.

Supported lipid bilayers (SLBs) are widely used as a model for studying membrane properties (phase separation, clustering, dynamics) and its interaction ...

Microfluidic Genipin Deposition Technique for Extended Culture of Micropatterned Vascular Muscular Thin Films

The chronic nature of vascular disease progression requires the development of experimental techniques that simulate physiologic and pathologic vascular behaviors on disease-relevant time scales. Previously, microcontact printing has been used to fabricate two-dimensional functional arterial mimics through patterning of extracellular matrix protein as guidance cues for tissue organization. Vascular muscular thin films utilized these mimics to assess functional contractility. However, the microcontact printing fabrication technique used typically incorporates hydrophobic PDMS substrates. As the tissue turns over the underlying extracellular matrix, new proteins must undergo a conformational change or denaturing in order to expose hydrophobic amino acid residues to the hydrophobic PDMS surfaces for attachment, resulting in altered matrix protein bioactivity, delamination, and death of the tissues.
Here, we present a microfluidic deposition technique for patterning of the crosslinker compound genipin. Genipin serves as an intermediary between patterned tissues and PDMS substrates, allowing cells to deposit newly-synthesized extracellular matrix protein onto a more hydrophilic surface and remain attached to the PDMS substrates. We also show that extracellular matrix proteins can be patterned directly onto deposited genipin, allowing dictation of engineered tissue structure. Tissues fabricated with this technique show high fidelity in both structural alignment and contractile function of vascular smooth muscle tissue in a vascular muscular thin film model. This technique can be extended using other cell types and provides the framework for future study of chronic tissue- and organ-level functionality.

We present a method for microfluidic deposition of patterned genipin and fibronectin on PDMS substrates, allowing extended viability of vascular smooth muscle cell-dense tissues. This tissue fabrication method is combined with previous vascular muscular thin film technology to measure vascular contractility over disease-relevant time courses.

The chronic nature of vascular disease progression requires the development of experimental techniques that simulate physiologic and pathologic vascular ...

Layer-by-layer Collagen Deposition in Microfluidic Devices for Microtissue Stabilization

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D culture of cells in collagen type I gels helps to stabilize cell morphology and function, which is necessary for creating microfluidic tissue models in microdevices. Translating traditional 3D culture techniques for tissue culture plates to microfluidic devices is often difficult because of the limited channel dimensions. In this method, we describe a technique for modifying native type I collagen to generate polycationic and polyanionic collagen solutions that can be used with layer-by-layer deposition to create ultrathin collagen assemblies on top of cells cultured in microfluidic devices. These thin collagen layers stabilize cell morphology and function, as shown using primary hepatocytes as an example cell, allowing for the long term culture of microtissues in microfluidic devices.

The creation of functional microtissues within microfluidic devices requires the stabilization of cell phenotypes by adapting traditional cell culture techniques to the limited spatial dimensions in microdevices. Modification of collagen allows the layer-by-layer deposition of ultrathin collagen assemblies that can stabilize primary cells, such as hepatocytes, as microfluidic tissue models.

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D ...

Proper Positioning and Restraint of a Rat Hind Limb for Focused High Resolution Imaging of Bone Micro-architecture Using In Vivo Micro-computed Tomography

The use of in vivo micro-computed tomography (&#181;CT) is a powerful tool which involves the non-destructive imaging of internal structures at high resolutions in live animal models. This allows for repeated imaging of the same rodent over time. This feature not only reduces the total number of rodents required in an experimental design and thereby reduces the inter-subject variation that can arise, but also allows researchers to assess longitudinal or life-long responses to an intervention. To acquire high quality images that can be processed and analyzed to more accurately quantify outcomes of bone micro-architecture, users of in vivo &#181;CT scanners must properly anesthetize the rat, and position and restrain the hind limb. To do this, it is imperative that the rat be anesthetized to a level of complete relaxation, and that pedal reflexes are lost. These guidelines may be modified for each individual rat, as the rate of isoflurane metabolism can vary depending on strain and body size. Proper technique for in vivo &#181;CT image acquisition enables accurate and consistent measurement of bone micro-architecture within and across studies.

Proper Positioning and Restraint of a Rat Hind Limb for Focused High Resolution Imaging of Bone Micro-architecture Using In Vivo Micro-computed Tomography

This paper instructs users of in vivo micro-computed tomography (&#181;CT) scanners how to anesthetize, correctly position and restrain the hind limb of a rat for minimal movement during high-resolution imaging of the tibia. The result is high quality images that can be processed to accurately quantify bone micro-architecture.

The use of in vivo micro-computed tomography (&#181;CT) is a powerful tool which involves the non-destructive imaging of internal structures at high ...