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....

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 rin....

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 defined by an .......

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

List of materials used in this article

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.