Fabrication of Zero Mode Waveguides for High Concentration Single Molecule Microscopy

Summary

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.

Abstract

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 100−200 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 100−200 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.

Introduction

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 transcription^1,2,3, translation^4,5,6, 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 100−200 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 μM to mM range¹⁰, biochemical reactions in single molecule studies are usually much slower than those in the cell. For example, protein synthesis occurs at 15−20  amino acids per second in E. coli^11,12, while most prokaryotic ribosomes in smFRET experiments translate at 0.1−1 amino acid per second¹³. In protein synthesis, crystal structures and smFRET on stalled ribosomes showed that transfer RNAs (tRNAs) fluctuate between ‘hybrid’ and ‘classical’ states before the tRNA-mRNA translocation step^14,15. 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 100−200 nm in diameter and 100−150 nm in depth¹⁷. Above a cutoff wavelength related to the size and shape of the wells (λ_c ≈ 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 waveguide^18,19. Although similar to TIR evanescent waves, ZMW evanescent waves have a shorter decay constant, resulting in 10−30 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 milling^25,26 or electron beam lithography (EBL) followed by plasma-etching^16,27. 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 “natural” lithography, which was first proposed in 1982 by Deckman and Dunsmuir^29,30, 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.

Protocol

NOTE: All steps can be completed in general lab space.

1. Glass coverslip cleaning

1. To provide a clean surface for evaporative deposition of colloidal particles, place 24 x 30 mm optical borosilicate glass coverslips (0.16−0.19 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.
2. Pour enough acetone in the staining jar to cover the coverslips, place the cover on, and sonicate for 10 min at 40 °C.
3. 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.
4. Repeat the acetone sonication (steps 1.2 and 1.3) once more.
5. Pour enough 200 mM KOH in the jar to cover the coverslips and sonicate, covered, for 20 min at 40 °C.
   NOTE: The KOH slightly etches the glass.
6. Rinse the coverslips with distilled H₂O 6 times.
7. Add ethanol to cover the coverslips, add the lid, and sonicate for 10 min at 40 °C.
8. Rinse the coverslips with distilled H₂O 3 times.
9. 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.

2. Evaporative deposition of polystyrene beads

1. To create the colloidal crystal mask for the ZMW array, centrifuge 50 µL of 1 µm diameter, non-functionalized polystyrene beads (2.5% w/v in water) at 15,000 x g, 25 °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.
2. 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.
3. Resuspend the beads from step 2.2 in 50 µL 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.
4. 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.
5. Place a hygrometer and a small electric fan centered behind the Petri dishes.
6. Record the starting relative humidity (RH) in the lab. Fill a 200 mL beaker with 150−200 mL of ~75 °C water and place it behind the fan.
7. 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).
8. Let the RH in the chamber rise to 70−75%, which typically takes 5−10 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).
9. When the RH reaches 70−75%, 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 25−26 °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.
10. Let the RH in the chamber continue to rise to 85%. At that point, record the RH in the humidity chamber and pipette 5 µL of the bead suspension onto the center of each coverslip.
11. Close the chamber and Petri dishes after each deposition to minimize loss of humidity. Aim to finish all 6 depositions within 2 min.
12. 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 70−75% RH after the deposition.
13. 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).

3. Bead annealing for reducing pore size in the colloidal crystal template

1. To provide a uniform temperature surface for annealing of the polystyrene beads, which narrows the inter-bead interstices and rounds the interstices’ corners, place a flat, milled aluminum plate on top of a standard ceramic hot plate.
2. Set the temperature of the hot plate to 107 °C, the glass transition temperature of polystyrene⁴⁰.
   NOTE: To obtain stable and accurate temperature, a thermocouple probe was held in a 2−3 mm wide and 4−5 mm deep hole in the aluminum plate.
3. 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).
4. 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.

4. Nanofabrication of aluminum zero mode waveguides using the colloidal crystal template

1. Using thermal or electron-beam evaporative deposition, deposit 300 nm of copper at 2 Å/s over the colloidal crystal template to generate posts in the interstices between the beads.
2. 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.
3. 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.
4. After the toluene incubation, rinse the templates once with chloroform and twice with ethanol. Handle coverslips carefully at this point because the delicate 200−300 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.
5. Using thermal or electron-beam evaporative deposition, deposit 3 nm of a titanium adhesion layer at 1 Å/s followed by 100−150 nm of aluminum at 4 Å/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).
6. 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.
7. 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.

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.

1. Using thermal or electron beam evaporative deposition, deposit 3 nm of a titanium adhesion layer at 1 Å/s followed by 300 nm of aluminum at 4 Å/s.
2. Remove excess metal on top of the beads by gently pressing the surface with tape. Slowly peel the tape to pull off the metal.
3. To dissolve the polystyrene beads, place the bead templates in toluene and soak overnight.
4. 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.
5. Using thermal or electron beam evaporative deposition, deposit 100−150 nm of gold at 5 Å/s around and on top of the aluminum posts.
6. To dissolve the metal posts, soak the coverslips in aluminum etchant (phosphoric acid-based; Table of Materials) for 1 h.
7. 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.

Results

The self-assembly of the polystyrene colloidal particles via evaporative sedimentation (steps 2.1−2.13) can produce a range of results since it requires control of the solvent evaporation rate. However, because the depositions are fast (10−15 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...

Discussion

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 2−3 min after deposition rather than 1−2 h as in previous methods^48,49. 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

Materials

Name	Company	Catalog Number	Comments
1. Glass Coverslip Cleaning
Acetone	Sigma	32201	1 L
Coplin glass staining jar	Fisher Scientific	08-817	Staining jar with 8 grooves and molded glass cover
Coverslips	VWR	48404-467	24 mm x 30 mm (No.1½, Rectangular)
Ethanol	Sigma	E7023	1 L
KOH	Sigma	30603	Potassium hydroxide
Petri dishes	Fisher Scientific	R80115TS	100 mm diameter, 15 mm deep
Sonicator	Branson	Z245143	Tabletop ultrasonic cleaner, 5510
2. Evaporative Deposition of Polystyrene Beads
Clear storage container	Fisher Scientific	50-110-8222	26 x 18 x 15 in.
Desk fan	O2Cool	FD05001A	Any small desk (~5 in.) fan will work
Glass beaker	Fisher Scientific	02-555-25B	250 mL
Humidity meter	Fisher Scientific	11-661-19
Microcentrifuge tubes	Fisher Scientific	21-402-903	1.5 mL
Polystyrene microspheres	Polysciences	18602-15	1.00 µm diameter, non-functionalized
Triton X-100 deturgent	Sigma	X100	100 mL
3. Bead Annealing for Reducing Pore Size in the Colloidal Crystal Template
Aluminum plate	Fisher Scientific	AA11062RY	Customized in-house to 14 cm x 14 cm
Ceramic hotplate	Fisher Scientific	HP88857100	13 x 8.2 x 3.8 in.
Temperature controller	McMaster-Carr	38615K71	Read temperature with thermocouple probe
Thermocouple probe	McMaster-Carr	9251T93	Type K, surface probe
4/5. Nanofabrication of Zero Mode Waveguides Using the Colloidal Crystal Template
Aluminum etchant	Transene	Type A
Aluminum pellets	Kurt J. Lesker	EVMAL40QXHB	For electron beam evaporation
Chloroform	Sigma	288306	1 L
Copper etchant	Transene	49-1
Copper pellets	Kurt J. Lesker	EVMCU40QXQA	For electron beam evaporation
Gold pellets	Kurt J. Lesker	EVMAUXX40G	For electron beam evaporation
Lens paper	Thorlabs	MC-5
Plasma cleaner	Harrick Plasma	PDC-32G
Scotch tape	Staples	MMM119
Thin film deposition system	Kurt J. Lesker	PVD-75	Tabletop thermal evaporation system will also work
Titanium pellets	Kurt J. Lesker	EVMTI45QXQA	For electron beam evaporation
Toluene	Sigma	244511	1 L
Representative Results
COMSOL Multiphysics Modeling Software	COMSOL, Inc.
Dual View spectral splitter	Photometrics, Inc.