This work was supported by NIH grants R01GM080376, R35GM118139, and NSF Center for Engineering MechanoBiology CMMI: 15-48571 to Y.E.G., and by an NIAID pre-doctoral NRSA fellowship F30AI114187 to R.M.J.

NOTE: All steps can be completed in general lab space.
1. Glass coverslip cleaning
<ol>
	<li>To provide a clean surface for evaporative deposition of colloidal particles, place 24 x 30 mm optical borosilicate glass coverslips (0.16&#8722;0.19 mm thickness) within the grooved inserts of a coplin glass staining jar for cleaning. 
	NOTE: Make sure the coverslips stand upright and are well-separated so that all surfaces are clearly exposed during the cleaning process.</li>
	<li>Pour enough acetone in the staining jar to cover the coverslips, place the cover on, and sonicate for 10 min at 40 &#176;C.</li>
	<li>Pour out the acetone and rinse the coverslips by filling the staining jar with distilled H2O and pouring out the water. Repeat 2 more times.</li>
	<li>Repeat the acetone sonication (steps 1.2 and 1.3) once more.</li>
	<li>Pour enough 200 mM KOH in the jar to cover the coverslips and sonicate, covered, for 20 min at 40 &#176;C. 
	NOTE: The KOH slightly etches the glass.</li>
	<li>Rinse the coverslips with distilled H2O 6 times.</li>
	<li>Add ethanol to cover the coverslips, add the lid, and sonicate for 10 min at 40 &#176;C.</li>
	<li>Rinse the coverslips with distilled H2O 3 times.</li>
	<li>Pick up each coverslip at the edge using gentle forceps and dry the coverslips with N2 gas. Touch only the edges of the coverslip. Place each of the dried, cleaned coverslips in an individual clean Petri dish.</li>
</ol>
2. Evaporative deposition of polystyrene beads
<ol>
	<li>To create the colloidal crystal mask for the ZMW array, centrifuge 50 &#181;L of 1 &#181;m diameter, non-functionalized polystyrene beads (2.5% w/v in water) at 15,000 x g, 25 &#176;C for 5 min. 
	NOTE: Before pipetting the beads, the stock solution should be briefly vortexed in case the beads have settled to the bottom of the bottle.</li>
	<li>Discard the supernatant, leaving as little water remaining as possible. 
	NOTE: Residual water can change the evaporation properties of the ethanol resuspension39, so removal of a small amount of beads in order to remove all of the water is acceptable.</li>
	<li>Resuspend the beads from step 2.2 in 50 &#181;L of 1:400 TritonX-100:ethanol solvent. Pipette up and down several times to thoroughly mix the beads with the solvent. 
	NOTE: TritonX-100:ethanol solvent should be sealed with paraffin film after use and prepared fresh once a month. The beads tend to adhere to the sides of a plastic vessel, such as a microcentrifuge tube, so pipette along the sides to ensure that all beads are resuspended.</li>
	<li>To set up a humidity chamber for deposition, place 6 Petri dishes, each with one coverslip, on a bench in a line with lids left slightly ajar. In each dish, move the coverslip to the open region so that the coverslips are exposed to the environment when the humidity is increased in the next step.</li>
	<li>Place a hygrometer and a small electric fan centered behind the Petri dishes.</li>
	<li>Record the starting relative humidity (RH) in the lab. Fill a 200 mL beaker with 150&#8722;200 mL of ~75 &#176;C water and place it behind the fan.</li>
	<li>Turn on the fan and cover the Petri dishes, fan, beaker, and hygrometer with an overturned, transparent plastic storage container (66 cm x 46 cm x 38 cm).</li>
	<li>Let the RH in the chamber rise to 70&#8722;75%, which typically takes 5&#8722;10 min. 
	NOTE: If the ambient lab RH is low (below ~50%), let the chamber reach a higher RH, but no higher than 80%, to compensate for loss of humidity during deposition (see below).</li>
	<li>When the RH reaches 70&#8722;75%, record the RH and lift up the plastic storage container slightly to quickly place covers on the Petri dishes, which prevents over-wetting of the coverslips. 
	NOTE: The temperature in the chamber will be slightly warmer than room temperature, typically 25&#8722;26 &#176;C, as a result of humidification. If moisture is visible on the coverslips, then the glass surfaces are too wet. A commercial glove box might simplify this part of the protocol.</li>
	<li>Let the RH in the chamber continue to rise to 85%. At that point, record the RH in the humidity chamber and pipette 5 &#181;L of the bead suspension onto the center of each coverslip.</li>
	<li>Close the chamber and Petri dishes after each deposition to minimize loss of humidity. Aim to finish all 6 depositions within 2 min.</li>
	<li>Record the RH in the chamber after the deposition. 
	NOTE: The RH after deposition will help gauge how fast humidity was lost during deposition, which depends on ambient lab conditions. For a typical successful run, the chamber will start at 85% RH prior to deposition and end at 70&#8722;75% RH after the deposition.</li>
	<li>Let the bead droplets spread and dry for 5 min. 
	NOTE: If the colloidal crystals have many holes or multilayered regions, then the chamber was likely too humid or dry, respectively. Adjust the relative humidity at which to close the Petri dishes and begin the depositions (see the results section for further discussion of optimization).</li>
</ol>
3. Bead annealing for reducing pore size in the colloidal crystal template
<ol>
	<li>To provide a uniform temperature surface for annealing of the polystyrene beads, which narrows the inter-bead interstices and rounds the interstices&#8217; corners, place a flat, milled aluminum plate on top of a standard ceramic hot plate.</li>
	<li>Set the temperature of the hot plate to 107 &#176;C, the glass transition temperature of polystyrene40. 
	NOTE: To obtain stable and accurate temperature, a thermocouple probe was held in a 2&#8722;3 mm wide and 4&#8722;5 mm deep hole in the aluminum plate.</li>
	<li>Place a coverslip containing the bead template on the hot aluminum plate and anneal for 20 s (see the discussion section for explanation of melting time).</li>
	<li>After heating, remove the coverslip from the aluminum plate and promptly place it on another room temperature aluminum surface to cool it. 
	NOTE: It is helpful to either have the coverslips hang slightly over the edge of the plate or mill shallow channels (see the accompanying video) into the plate to facilitate pickup of the coverslips.</li>
</ol>
4. Nanofabrication of aluminum zero mode waveguides using the colloidal crystal template
<ol>
	<li>Using thermal or electron-beam evaporative deposition, deposit 300 nm of copper at 2 &#197;/s over the colloidal crystal template to generate posts in the interstices between the beads.</li>
	<li>Remove excess metal on top of the beads by gently pressing the surface with tape. Slowly peel the tape to pull off the metal. 
	NOTE: Some small patches of reflective excess metal may remain after the tape pull, and these can often be removed by a stream of N2 gas. If substantial patches of reflective excess metal remain after the tape pull, try soaking the templates in toluene for 2 h to partially dissolve the polystyrene beads. Wash the coverslips with distilled water, dry with N2, and repeat the tape pull. The additional soak should not completely dissolve the beads, as the beads help protect the posts from damage during the tape pull.</li>
	<li>To dissolve the polystyrene beads, place the bead templates in toluene and soak overnight. 
	CAUTION: Toluene fumes may be toxic. Work with toluene under a well-ventilated hood and wear personal protective equipment, including gloves, safety glasses, and a lab coat. Toluene should be stored in ventilated cabinets designated for flammable liquids.</li>
	<li>After the toluene incubation, rinse the templates once with chloroform and twice with ethanol. Handle coverslips carefully at this point because the delicate 200&#8722;300 nm tall metal posts are now exposed. Dry the templates with N2 and remove residual polymer and contaminants in an oxygen plasma cleaner for 30 min. 
	CAUTION: Chloroform fumes may be toxic. Work with chloroform under a well-ventilated hood and wear personal protective equipment, including gloves, safety glasses, and a lab coat. Chloroform should be stored in ventilated cabinets away from other flammable solvents.</li>
	<li>Using thermal or electron-beam evaporative deposition, deposit 3 nm of a titanium adhesion layer at 1 &#197;/s followed by 100&#8722;150 nm of aluminum at 4 &#197;/s around and on top of the copper posts. 
	NOTE: One can use thicker cladding to obtain deeper guides and better attenuation of background fluorescence, but this also decreases the yield after exposing and dissolving the posts in the next step (see the discussion section).</li>
	<li>To dissolve the metal posts, soak the coverslips in copper etchant (citric acid-based; Table of Materials) for 2 h. 
	CAUTION: Metal etchant can cause skin burns. Work with etchants under a well-ventilated hood and wear protective equipment. Wash hands thoroughly after handling. Metal etchant should be stored in ventilated cabinets designated for corrosive liquids.</li>
	<li>Rinse the coverslips with distilled water, dry with N2, and gently buff the surface of the metal cladding with lens paper to expose any posts that are still covered in cladding. Place the coverslips back in copper etchant for another 2 h, then rinse again with distilled water and dry with N2. 
	NOTE: ZMW slides should be stored in covered, clean Petri dishes to keep them free of contaminants.</li>
</ol>
5. Nanofabrication of gold zero mode waveguides using the colloidal crystal template
NOTE: The method to fabricate gold ZMWs (Supplemental Figure 1), which mirrors the protocol to fabricate aluminum ZMWs, is provided in this section.
<ol>
	<li>Using thermal or electron beam evaporative deposition, deposit 3 nm of a titanium adhesion layer at 1 &#197;/s followed by 300 nm of aluminum at 4 &#197;/s.</li>
	<li>Remove excess metal on top of the beads by gently pressing the surface with tape. Slowly peel the tape to pull off the metal.</li>
	<li>To dissolve the polystyrene beads, place the bead templates in toluene and soak overnight.</li>
	<li>After the toluene incubation, rinse the templates once with chloroform and twice with ethanol. Dry the templates with N2 and remove residual polymer contaminants in an oxygen plasma cleaner for 30 min.</li>
	<li>Using thermal or electron beam evaporative deposition, deposit 100&#8722;150 nm of gold at 5 &#197;/s around and on top of the aluminum posts.</li>
	<li>To dissolve the metal posts, soak the coverslips in aluminum etchant (phosphoric acid-based; Table of Materials) for 1 h.</li>
	<li>Rinse the coverslips with distilled water, dry with N2, and gently buff the surface of the metal cladding with lens paper to expose any posts that are still covered in cladding. Place the coverslips back in aluminum etchant for 1 h, then rinse again with distilled water and dry with N2. 
	NOTE: ZMW slides should be stored in covered, clean Petri dishes.</li>
</ol>

The authors have nothing to disclose.

For the colloidal self-assembly (protocol section 2), the use of ethanol rather than water as the suspension solvent speeds the evaporation process so that templates are ready in 2&#8722;3 min after deposition rather than 1&#8722;2 h as in previous methods48,49. The evaporative sedimentation protocol presented here is also simpler than previous sedimentation protocols that require controlling surface tilt, temperature, and air volume above the suspension<sup clas...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/61154">Fabrication of Zero Mode Waveguides for High Concentration Single Molecule Microscopy</a>. A figure was updated.
Figure 3 was updated from:
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61154/61154fig3.jpg" /> 
Figure 3: Representative results from evaporative deposition of colloids. (A) Example of optimal colloid deposition. (B) Example of an acceptable colloid deposition in which conditions were more humid (80% RH) than ideal. Holes in the crystal monolayer are apparent. (C) Example of an acceptable colloid deposition in which conditions were drier (65% RH) than optimal. The monolayer regions are slightly translucent while multilayered areas are white and opaque (perimeter and streaks inward). (D) A colloidal crystal illuminated with white light to highlight the rainbow diffraction from the crystals. (E) AFM image (tapping probe AFM in air) of a monolayer of hexagonally packed polystyrene beads from a successful colloid deposition (scale bar = 10 &#181;m). (F) Expanded AFM image of packed beads (scale bar = 2 &#181;m). <a href="https://www.jove.com/files/ftp_upload/61154/61154fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
to:
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61154/61154fig3v2.jpg" /> 
Figure 3: Representative results from evaporative deposition of colloids. (A) Example of optimal colloid deposition. (B) Example of an acceptable colloid deposition in which conditions were more humid (80% RH) than ideal. Holes in the crystal monolayer are apparent. (C) Example of an acceptable colloid deposition in which conditions were drier (65% RH) than optimal. The monolayer regions are slightly translucent while multilayered areas are white and opaque (perimeter and streaks inward). (D) A colloidal crystal illuminated with white light to highlight the rainbow diffraction from the crystals. (E) AFM image (tapping probe AFM in air) of a monolayer of hexagonally packed polystyrene beads from a successful colloid deposition (scale bar = 10 &#181;m). (F) Expanded AFM image of packed beads (scale bar = 2 &#181;m). <a href="https://www.jove.com/files/ftp_upload/61154/61154fig3v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

An erratum was issued for:&#160;<a href="https://www.jove.com/t/61154">Fabrication of Zero Mode Waveguides for High Concentration Single Molecule Microscopy</a>. A figure was updated.

Erratum: Fabrication of Zero Mode Waveguides for High Concentration Single Molecule Microscopy

Single-molecule techniques such as single molecule fluorescence resonance energy transfer (smFRET) or single molecule fluorescence correlation spectroscopy (FCS) are powerful tools for molecular biophysics, allowing the study of dynamic movements, conformations, and interactions of individual biomolecules in processes such as transcription1,2,3, translation4,5,6, and many others7. For smFRET, total internal reflection fluorescence (TIRF) microscopy is a common method because many tethered molecules may be followed over time, and the evanescent wave generated by TIR is limited to a 100&#8722;200 nm region adjacent to the coverslip8. However, even with this restriction on excitation volume, fluorophores of interest still need to be diluted to pM or nM ranges in order to detect single molecule signals above background fluorecence9. Since the Michaelis-Menten constants of cellular enzymes are typically in the &#956;M to mM range10, biochemical reactions in single molecule studies are usually much slower than those in the cell. For example, protein synthesis occurs at 15&#8722;20 &#160;amino acids per second in E. coli11,12, while most prokaryotic ribosomes in smFRET experiments translate at 0.1&#8722;1 amino acid per second13. In protein synthesis, crystal structures and smFRET on stalled ribosomes showed that transfer RNAs (tRNAs) fluctuate between &#8216;hybrid&#8217; and &#8216;classical&#8217; states before the tRNA-mRNA translocation step14,15. However, when physiological concentrations of the translocation GTPase factor, EF-G, was present, a different conformation, intermediate between the hybrid and classical states, was observed in smFRET6. Studying dynamic molecular processes at rates and concentrations similar to those in the cell is important, but remains a technical challenge.
A strategy to increase the fluorescent substrate concentration is the use of metal-based, sub-visible wavelength apertures, called zero mode waveguides (ZMWs), to generate confined excitation fields that selectively excite biomolecules localized within the apertures16 (Figure 1). The apertures are typically 100&#8722;200 nm in diameter and 100&#8722;150 nm in depth17. Above a cutoff wavelength related to the size and shape of the wells (&#955;c &#8776; 2.3 times the diameter for circular waveguides with water as the dielectric medium18), no propagating modes are allowed in the waveguide, hence the term zero mode waveguides. However, an oscillating electromagnetic field, termed an evanescent wave, exponentially decaying in intensity still tunnels a short distance into the waveguide18,19. Although similar to TIR evanescent waves, ZMW evanescent waves have a shorter decay constant, resulting in 10&#8722;30 nm effective excitation region within the waveguide. At micromolar concentrations of fluorescently labeled ligands, only one or a few molecules are simultaneously present within the excitation region. This restriction of the excitation volume and consequent reduction of background fluorescence enables fluorescence imaging of single molecules at biologically relevant concentrations. This has been applied to many systems20, including FCS measurements of single protein diffusion21, single molecule FRET measurements of low-affinity ligand-protein22 and protein-protein interactions23, and spectro-electrochemical measurements of single molecular turnover events24.
ZMWs have been produced by directly patterning a metal layer using ion beam milling25,26 or electron beam lithography (EBL) followed by plasma-etching16,27. These maskless lithography methods create waveguides in series and typically require access to specialized nanofabrication facilities, preventing widespread adoption of ZMW technology. Another method, ultraviolet nanoimprint lithography lift-off28, uses a quartz slide mold to press an inverse ZMW template onto a resist film like a stamp. While this method is more streamlined, it still requires EBL for fabrication of the quartz mold. This article presents the protocol for a simple and inexpensive templated fabrication method that does not require EBL or ion-beam milling and is based on close-packing of nanospheres to form a lithographic mask.
Nanosphere or &#8220;natural&#8221; lithography, which was first proposed in 1982 by Deckman and Dunsmuir29,30, uses the self-assembly of monodisperse colloidal particles, ranging from tens of nanometers to tens of micrometers31, to create templates for surface patterning via etching and/or deposition of materials. The two-dimensional (2D) or three-dimensional (3D) extended periodic arrays of colloidal particles, referred to as colloidal crystals, are characterized by a bright iridescence from scattering and diffraction32. Although less widely used than electron-beam or photolithography, this masking methodology is simple, low cost, and easily scaled down to create feature sizes below 100 nm.
Directing the self-assembly of colloidal particles determines the success of using colloidal crystals as masks for surface patterning. If the size and shape of particles are homogeneous, colloidal particles can be readily self-assembled with hexagonal packing, driven by entropic depletion33. Water evaporation after drop-coating is an effective route to sediment the colloidal particles, although other methods include dip-coating34, spin coating35, electrophoretic deposition36, and consolidation at an air-water interface37. The protocol presented below is based on the evaporation sedimentation method, which was the simplest to implement. The triangular interstices between close-packed polystyrene beads form openings in which to plate a sacrificial metal, forming posts (Figure 2 and Supplemental Figure 1). Brief annealing of the beads before this step adjusts the shape and diameter of these posts. The beads are removed, a final metal layer is deposited around the posts, and then the posts are removed. After the two metal deposition steps onto the colloidal nanomask, removal of the intermediate posts, and surface chemistry modification for passivation and tethering, ZMW arrays are ready to use for single molecule imaging. More extensive characterization of the ZMW optical properties after fabrication can found in an accompanying article38. Besides a thermal evaporator for vapor deposition of the metals, no specialized tools are required.

<table border="0" cellpadding="0" cellspacing="0" style="width:667px;" width="668">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">1. Glass Coverslip Cleaning</td>
			<td style="width:103px;"></td>
			<td style="width:119px;"></td>
			<td style="width:231px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Acetone</td>
			<td style="width:103px;">Sigma</td>
			<td style="width:119px;">32201</td>
			<td>1 L</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Coplin glass staining jar</td>
			<td style="width:103px;">Fisher Scientific</td>
			<td style="width:119px;">08-817</td>
			<td>Staining jar with 8 grooves and molded glass cover</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Coverslips</td>
			<td style="width:103px;">VWR</td>
			<td style="width:119px;">48404-467</td>
			<td>24 mm x 30 mm (No.1&#189;, Rectangular)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Ethanol</td>
			<td style="width:103px;">Sigma</td>
			<td style="width:119px;">E7023</td>
			<td>1 L</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">KOH</td>
			<td style="width:103px;">Sigma</td>
			<td style="width:119px;">30603</td>
			<td>Potassium hydroxide</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Petri dishes</td>
			<td style="width:103px;">Fisher Scientific</td>
			<td style="width:119px;">R80115TS</td>
			<td>100 mm diameter, 15 mm deep</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Sonicator</td>
			<td style="width:103px;">Branson</td>
			<td style="width:119px;">Z245143</td>
			<td>Tabletop ultrasonic cleaner, 5510</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">2. Evaporative Deposition of Polystyrene Beads</td>
			<td style="width:103px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Clear storage container</td>
			<td style="width:103px;">Fisher Scientific</td>
			<td style="width:119px;">50-110-8222</td>
			<td>26 x 18 x 15 in.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Desk fan</td>
			<td style="width:103px;">O2Cool</td>
			<td style="width:119px;">FD05001A</td>
			<td>Any small desk (~5 in.) fan will work</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Glass beaker</td>
			<td style="width:103px;">Fisher Scientific</td>
			<td style="width:119px;">02-555-25B</td>
			<td>250 mL</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Humidity meter</td>
			<td style="width:103px;">Fisher Scientific</td>
			<td style="width:119px;">11-661-19</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Microcentrifuge tubes</td>
			<td style="width:103px;">Fisher Scientific</td>
			<td style="width:119px;">21-402-903</td>
			<td>1.5 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Polystyrene microspheres</td>
			<td style="width:103px;">Polysciences</td>
			<td style="width:119px;">18602-15</td>
			<td>1.00 &#181;m diameter, non-functionalized</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Triton X-100 deturgent</td>
			<td style="width:103px;">Sigma</td>
			<td style="width:119px;">X100</td>
			<td>100 mL</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:215px;">3. Bead Annealing for Reducing Pore Size in the Colloidal Crystal Template</td>
			<td style="width:103px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Aluminum plate</td>
			<td style="width:103px;">Fisher Scientific</td>
			<td style="width:119px;">AA11062RY</td>
			<td>Customized in-house to 14 cm x 14 cm</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Ceramic hotplate</td>
			<td style="width:103px;">Fisher Scientific</td>
			<td style="width:119px;">HP88857100</td>
			<td>13 x 8.2 x 3.8 in.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Temperature controller</td>
			<td style="width:103px;">McMaster-Carr</td>
			<td style="width:119px;">38615K71</td>
			<td>Read temperature with thermocouple probe</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Thermocouple probe</td>
			<td style="width:103px;">McMaster-Carr</td>
			<td style="width:119px;">9251T93</td>
			<td>Type K, surface probe</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:215px;">4/5. Nanofabrication of Zero Mode Waveguides Using the Colloidal Crystal Template</td>
			<td style="width:103px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Aluminum etchant</td>
			<td style="width:103px;">Transene</td>
			<td style="width:119px;">Type A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Aluminum pellets</td>
			<td style="width:103px;">Kurt J. Lesker</td>
			<td style="width:119px;">EVMAL40QXHB</td>
			<td>For electron beam evaporation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Chloroform</td>
			<td style="width:103px;">Sigma</td>
			<td style="width:119px;">288306</td>
			<td>1 L</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Copper etchant</td>
			<td style="width:103px;">Transene</td>
			<td style="width:119px;">49-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Copper pellets</td>
			<td style="width:103px;">Kurt J. Lesker</td>
			<td style="width:119px;">EVMCU40QXQA</td>
			<td>For electron beam evaporation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Gold pellets</td>
			<td style="width:103px;">Kurt J. Lesker</td>
			<td style="width:119px;">EVMAUXX40G</td>
			<td>For electron beam evaporation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Lens paper</td>
			<td style="width:103px;">Thorlabs</td>
			<td style="width:119px;">MC-5</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Plasma cleaner</td>
			<td style="width:103px;">Harrick Plasma</td>
			<td style="width:119px;">PDC-32G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Scotch tape</td>
			<td style="width:103px;">Staples</td>
			<td style="width:119px;">MMM119</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Thin film deposition system</td>
			<td style="width:103px;">Kurt J. Lesker</td>
			<td style="width:119px;">PVD-75</td>
			<td>Tabletop thermal evaporation system will also work</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Titanium pellets</td>
			<td style="width:103px;">Kurt J. Lesker</td>
			<td style="width:119px;">EVMTI45QXQA</td>
			<td>For electron beam evaporation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Toluene</td>
			<td style="width:103px;">Sigma</td>
			<td style="width:119px;">244511</td>
			<td>1 L</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Representative Results</td>
			<td style="width:103px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">COMSOL Multiphysics Modeling Software</td>
			<td>COMSOL, Inc.</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Dual View spectral splitter</td>
			<td style="width:103px;">Photometrics, Inc.</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

fabrication of zero mode waveguides for high concentration single molecule microscopy

In single molecule fluorescence enzymology, background fluorescence from labeled substrates in solution often limits fluorophore concentration to pico- to nanomolar ranges, several orders of magnitude less than many physiological ligand concentrations. Optical nanostructures called zero mode waveguides (ZMWs), which are 100&#8722;200 nm in diameter apertures fabricated in a thin conducting metal such as aluminum or gold, allow imaging of individual molecules at micromolar concentrations of fluorophores by confining visible light excitation to zeptoliter effective volumes. However, the need for expensive and specialized nanofabrication equipment has precluded the widespread use of ZMWs. Typically, nanostructures such as ZMWs are obtained by direct writing using electron beam lithography, which is sequential and slow. Here, colloidal, or nanosphere, lithography is used as an alternative strategy to create nanometer-scale masks for waveguide fabrication. This report describes the approach in detail, with practical considerations for each phase. The method allows thousands of aluminum or gold ZMWs to be made in parallel, with final waveguide diameters and depths of 100&#8722;200 nm. Only common lab equipment and a thermal evaporator for metal deposition are required. By making ZMWs more accessible to the biochemical community, this method can facilitate the study of molecular processes at cellular concentrations and rates.

The self-assembly of the polystyrene colloidal particles via evaporative sedimentation (steps 2.1&#8722;2.13) can produce a range of results since it requires control of the solvent evaporation rate. However, because the depositions are fast (10&#8722;15 min per round), the procedure can be quickly optimized for different ambient lab conditions. Figure 3A shows a well-formed colloidal template after deposition and evaporation. Macroscopically, the region of beads is circular, with borders de...

Watch this Scientific Journal Video about Fabrication of Zero Mode Waveguides for High Concentration Single Molecule Microscopy at JoVE.com

Fabrication of Zero Mode Waveguides for High Concentration Single Molecule Microscopy

Described here is a nanosphere lithography method for parallel fabrication of zero mode waveguides, which are arrays of nanoapertures in a metal-clad glass microscopy coverslip for single molecule imaging at nano- to micromolar concentrations of fluorophores. The method takes advantage of colloidal crystal self-assembly to create a waveguide template.

In single molecule fluorescence enzymology, background fluorescence from labeled substrates in solution often limits fluorophore concentration to pico- to ...

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Research

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Bioengineering

7.9K Views.  University of Pennsylvania. Described here is a nanosphere lithography method for parallel fabrication of zero mode waveguides, which are arrays of nanoapertures in a metal-clad glass microscopy coverslip for single molecule imaging at nano- to micromolar concentrations of fluorophores. The method takes advantage of colloidal crystal self-assembly to create a waveguide template.

Video: Fabrication of Zero Mode Waveguides for High Concentration Single Molecule Microscopy

Low-Cost Cryo-Light Microscopy Stage Fabrication for Correlated Light/Electron Microscopy

The coupling of cryo-light microscopy (cryo-LM) and cryo-electron microscopy (cryo-EM) poses a number of advantages for understanding cellular 
dynamics and ultrastructure. First, cells can be imaged in a near native environment for both techniques. Second, due to the vitrification process, samples are
 preserved by rapid physical immobilization rather than slow chemical fixation. Third, imaging the same sample with both cryo-LM and cryo-EM provides correlation of
 data from a single cell, rather than a comparison of "representative samples". While these benefits are well known from prior studies, the widespread use of correlative 
 cryo-LM and cryo-EM remains limited due to the expense and complexity of buying or building a suitable cryogenic light microscopy stage. Here we demonstrate the 
 assembly, and use of an inexpensive cryogenic stage that can be fabricated in any lab for less than $40 with parts found at local hardware and grocery stores. This 
 cryo-LM stage is designed for use with reflected light microscopes that are fitted with long working distance air objectives. For correlative cryo-LM and cryo-EM 
 studies, we adapt the use of carbon coated standard 3-mm cryo-EM grids as specimen supports. After adsorbing the sample to the grid, previously established protocols 
 for vitrifying the sample and transferring/handling the grid are followed to permit multi-technique imaging. As a result, this setup allows any laboratory with a 
 reflected light microscope to have access to direct correlative imaging of frozen hydrated samples.

We demonstrate the fabrication of a low-cost cryogenic stage designed to fit most reflected light microscopes. This lab-built cryogenic stage enables efficient and reliable correlative imaging between cryo-light and cryo-electron microscopy.

The coupling of cryo-light microscopy (cryo-LM) and cryo-electron microscopy (cryo-EM) poses a number of advantages for understanding cellular 
dynamics ...

Multiplexed Single-molecule Force Proteolysis Measurements Using Magnetic Tweezers

The generation and detection of mechanical forces is a ubiquitous aspect of cell physiology, with direct relevance to cancer metastasis1, atherogenesis2 and wound healing3. In each of these examples, cells both exert force on their surroundings and simultaneously enzymatically remodel the extracellular matrix (ECM). The effect of forces on ECM has thus become an area of considerable interest due to its likely biological and medical importance4-7.
	Single molecule techniques such as optical trapping8, atomic force microscopy9, and magnetic tweezers10,11 allow researchers to probe the function of enzymes at a molecular level by exerting forces on individual proteins. Of these techniques, magnetic tweezers (MT) are notable for their low cost and high throughput. MT exert forces in the range of ~1-100 pN and can provide millisecond temporal resolution, qualities that are well matched to the study of enzyme mechanism at the single-molecule level12. Here we report a highly parallelizable MT assay to study the effect of force on the proteolysis of single protein molecules. We present the specific example of the proteolysis of a trimeric collagen peptide by matrix metalloproteinase 1 (MMP-1); however, this assay can be easily adapted to study other substrates and proteases.

In this article we describe the use of magnetic tweezers to study the effect of force on enzymatic proteolysis at the single molecule level in a highly parallelizable manner.

The  generation and detection of mechanical forces is a ubiquitous aspect of cell physiology,  with direct relevance to cancer metastasis1, atherogenesis2 ...

Fabrication of Carbon Nanotube High-Frequency Nanoelectronic Biosensor for Sensing in High Ionic Strength Solutions

The unique electronic properties and high surface-to-volume ratios of single-walled carbon nanotubes (SWNT) and semiconductor nanowires (NW) 1-4 make them good candidates for high sensitivity biosensors. When a charged molecule binds to such a sensor surface, it alters the carrier density5 in the sensor, resulting in changes in its DC conductance. However, in an ionic solution a charged surface also attracts counter-ions from the solution, forming an electrical double layer (EDL). This EDL effectively screens off the charge, and in physiologically relevant conditions ~100 millimolar (mM), the characteristic charge screening length (Debye length) is less than a nanometer (nm). Thus, in high ionic strength solutions, charge based (DC) detection is fundamentally impeded6-8.
We overcome charge screening effects by detecting molecular dipoles rather than charges at high frequency, by operating carbon nanotube field effect transistors as high frequency mixers9-11. At high frequencies, the AC drive force can no longer overcome the solution drag and the ions in solution do not have sufficient time to form the EDL. Further, frequency mixing technique allows us to operate at frequencies high enough to overcome ionic screening, and yet detect the sensing signals at lower frequencies11-12. Also, the high transconductance of SWNT transistors provides an internal gain for the sensing signal, which obviates the need for external signal amplifier.
Here, we describe the protocol to (a) fabricate SWNT transistors, (b) functionalize biomolecules to the nanotube13, (c) design and stamp a poly-dimethylsiloxane (PDMS) micro-fluidic chamber14 onto the device, and (d) carry out high frequency sensing in different ionic strength solutions11.

We describe the device fabrication and measurement protocol for carbon nanotube based high frequency biosensors. The high frequency sensing technique mitigates the fundamental ionic (Debye) screening effect and allows nanotube biosensor to be operated in high ionic strength solutions where conventional electronic biosensors fail. Our technology provides a unique platform for point-of-care (POC) electronic biosensors operating in physiologically relevant conditions.

The unique electronic properties and high surface-to-volume ratios of single-walled carbon nanotubes (SWNT) and semiconductor nanowires (NW) 1-4 make them ...

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

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

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

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

Investigating Receptor-ligand Systems of the Cellulosome with AFM-based Single-molecule Force Spectroscopy

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the enzymes onto the noncatalytic scaffold protein is directed by interactions among a family of related receptor-ligand pairs comprising interacting cohesin and dockerin modules. The extremely strong binding between cohesin and dockerin modules results in dissociation constants in the low picomolar to nanomolar range, which may hamper accurate off-rate measurements with conventional bulk methods. Single-molecule force spectroscopy (SMFS) with the atomic force microscope measures the response of individual biomolecules to force, and in contrast to other single-molecule manipulation methods (i.e.&#160;optical tweezers), is optimal for studying high-affinity receptor-ligand interactions because of its ability to probe the high-force regime (&#62;120 pN). Here we present our complete protocol for studying cellulosomal protein assemblies at the single-molecule level. Using a protein topology derived from the native cellulosome, we worked with enzyme-dockerin and carbohydrate binding module-cohesin (CBM-cohesin) fusion proteins, each with an accessible free thiol group at an engineered cysteine residue. We present our site-specific surface immobilization protocol, along with our measurement and data analysis procedure for obtaining detailed binding parameters for the high-affinity complex. We demonstrate how to quantify single subdomain unfolding forces, complex rupture forces, kinetic off-rates, and potential widths of the binding well. The successful application of these methods in characterizing the cohesin-dockerin interaction responsible for assembly of multidomain cellulolytic complexes is further described.

Cellulosomes are multienzyme complexes designed for digesting cellulose. AFM-based SMFS was used to study the mechanical properties and folding configuration of cellulosome-associated protein assemblies. We present a complete workflow for protein immobilization, data acquisition, and data analysis to study the interactions of individual receptor-ligand complexes involved in cellulosome assembly.

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of 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., ...

Ordering Single Cells and Single Embryos in 3D Confinement: A New Device for High Content Screening

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening based on cells in such environments have therefore serious limitations: cell organelles show extreme phenotypes, cell morphologies and sizes are heterogeneous and/or specific cell organelles cannot be properly visualized. In addition, cells in vivo are located in a 3D environment; in this situation, cells show different phenotypes mainly because of their interaction with the surrounding extracellular matrix of the tissue. In order to standardize and generate order of single cells in a physiologically-relevant 3D environment for cell-based assays, we report here the microfabrication and applications of a device for in vitro 3D cell culture. This device consists of a 2D array of microcavities (typically 105 cavities/cm2), each filled with single cells or embryos. Cell position, shape, polarity and internal cell organization become then normalized showing a 3D architecture. We used replica molding to pattern an array of microcavities, &#8216;eggcups&#8217;, onto a thin polydimethylsiloxane (PDMS) layer adhered on a coverslip. Cavities were covered with fibronectin to facilitate adhesion. Cells were inserted by centrifugation. Filling percentage was optimized for each system allowing up to 80%. Cells and embryos viability was confirmed. We applied this methodology for the visualization of cellular organelles, such as nucleus and Golgi apparatus, and to study active processes, such as the closure of the cytokinetic ring during cell mitosis. This device allowed the identification of new features, such as periodic accumulations and inhomogeneities of myosin and actin during the cytokinetic ring closure and compacted phenotypes for Golgi and nucleus alignment. We characterized the method for mammalian cells, fission yeast, budding yeast, C. elegans with specific adaptation in each case. Finally, the characteristics of this device make it particularly interesting for drug screening assays and personalized medicine.

We report a device and a new method to study cells and embryos. Single cells are precisely ordered in microcavity arrays. Their 3D confinement is a step towards 3D environments encountered in physiological conditions and allows organelle orientation. By controlling cell shape, this setup minimizes variability reported in standard assays.

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening ...

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 ...

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background in optics and laser physics to a discipline that is now enjoying vivid attention by life-scientists of all venues 1. This is because single molecule imaging has the unique potential to reveal protein behavior in situ in living cells and uncover cellular organization with unprecedented resolution below the diffraction limit of visible light 2. Glass-supported planar lipid bilayers (SLBs) are a powerful tool to bring cells otherwise growing in suspension in close enough proximity to the glass slide so that they can be readily imaged in noise-reduced Total Internal Reflection illumination mode 3,4. They are very useful to study the protein dynamics in plasma membrane-associated events as diverse as cell-cell contact formation, endocytosis, exocytosis and immune recognition. Simple procedures are presented how to generate highly mobile protein-functionalized SLBs in a reproducible manner, how to determine protein mobility within and how to measure protein densities with the use of single molecule detection. It is shown how to construct a cost-efficient single molecule microscopy system with TIRF illumination capabilities and how to operate it in the experiment.

Preparation of protein-functionalized planar glass-supported lipid bilayers, determination of protein mobility within and measurement of protein densities is shown here. A roadmap to building a noise-reduced Total Internal Reflection microscope is outlined, which allows visualizing single bilayer-resident fluorochromes with high spatiotemporal resolution.

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background ...

Label-free Single Molecule Detection Using Microtoroid Optical Resonators

Detecting small concentrations of molecules down to the single molecule limit has impact on areas such as early detection of disease, and fundamental studies on the behavior of molecules. Single molecule detection techniques commonly utilize labels such as fluorescent tags or quantum dots, however, labels are not always available, increase cost and complexity, and can perturb the events being studied. Optical resonators have emerged as a promising means to detect single molecules without the use of labels. Currently the smallest particle detected by a non-plasmonically-enhanced bare optical resonator system in solution is a 25 nm polystyrene sphere1. We have developed a technique known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER) that can surpass this limit and achieve label-free single molecule detection in aqueous solution2. As signal strength scales with particle volume, our work represents a &#62; 100x improvement in the signal to noise ratio (SNR) over the current state of the art. Here the procedures behind FLOWER are presented in an effort to increase its usage in the field.

We have developed a label-free biosensing system based on optical resonator technology known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER) that is capable of detecting single molecules in solution. Here the procedures behind this work are described and presented.

Detecting small concentrations of molecules down to the single molecule limit has impact on areas such as early detection of disease, and fundamental ...