Fabrication of Zero Mode Waveguides for High Concentration Single Molecule Microscopy
May 12th, 2020
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.
The nanosphere lithography protocol presented here for fabricating zero mode waveguides is an accessible, low cost method that does not require any specialized fabrication tools or facilities. This method will be useful for single molecule biophysics and allow more researchers to perform single molecule experiments at biologically relevant concentrations of fluorescent reagents. To create a clean surface for evaporative deposition of colloidal particles, place optical borosilicate glass coverslips in the grooved inserts of a Coplin glass staining jar.
Fill the staining jar with enough acetone to cover the coverslips and cover the jar. Sonicate the jar for 10 minutes at 40 degrees Celsius. Pour out the acetone and rinse the coverslips three times with distilled water.
After repeating the sonication and acetone, fill the jar with enough potassium hydroxide to cover the coverslips. Sonicate the jar again, covered, for 20 minutes at 40 degrees Celsius. Rinse the coverslips with distilled water six times, then sonicate them in ethanol for 10 minutes at 40 degrees Celsius.
After washing the coverslips three times with distilled water, gently pick up each coverslip at the edge using forceps. Dry each coverslip with nitrogen gas and place each clean, dry coverslip and its own Petri dish. Centrifuge 50 microliters of one micron non-functionalized polystyrene beads.
Discard the supernatant, leaving as little water remaining as possible. Resuspend the beads in 50 microliters of solvent, then mix thoroughly by pipetting. To set up a humidity chamber for deposition, arrange six Petri dishes in a row.
Place one coverslip in each Petri dish and leave the lids slightly ajar with the coverslips positioned to be exposed to the environment. Center a hygrometer and a small electric fan behind the Petri dishes, then record the relative humidity in the lab. Fill a 200 milliliter beaker with 150 to 200 milliliters of water at approximately 75 degrees Celsius and place the beaker behind the fan.
Turn on the fan and then cover the Petri dishes, fan, beaker, and hygrometer with an overturned, transparent plastic storage container. When the relative humidity reaches 70 to 75%lift the plastic storage container slightly and quickly close the lids of the Petri dishes to prevent overwetting of the coverslips. When the relative humidity in the chamber reaches 85%pipette five microliters of the bead suspension onto the center of each coverslip.
Cover each Petri dish immediately after deposition and keep everything covered the storage container between depositions. Bead depositions should be completed as quickly as possible to minimize the loss of humidity. If depositions are too dry or wet, chamber humidity is the most important variable to optimize.
When all six depositions are complete, record the relative humidity in the chamber and let the bead droplets spread and dry for five minutes. To provide a uniform temperature surface for annealing of the polystyrene beads, place a flat, milled aluminum plate on top of a standard ceramic hot plate and set the temperature of the hot plate to 107 degrees Celsius, the glass transition temperature of polystyrene. Place a coverslip containing the bead template on the hot aluminum plate.
After annealing it for 20 seconds, remove the coverslip from the aluminum plate and promptly place it on a room temperature aluminum surface to cool. Using thermal evaporative deposition, deposit 300 nanometers of copper at two angstroms per second over coverslips containing the bead templates. This will generate posts in the interstices between the beads.
To remove the excess copper from the beads, gently press a piece of tape onto the surface and then slowly peel off the tape. Dissolve the polystyrene beads by placing the coverslips in toleune and leaving them overnight. Handling the cover slips carefully, rinse them once with chloroform and twice with ethanol.
Dry the templates with nitrogen. Place them in an oxygen plasma cleaner for 30 minutes to remove residual polymer and contaminants. Using thermal evaporative deposition, deposit three nanometers of a titanium adhesion layer at one angstrom per second, then deposit 100 to 150 nanometers of aluminum at four angstroms per second around and on top of the copper posts.
Ensure that the samples are not rotating during deposition as is automatically done in some deposition units. To dissolve the copper posts, soak the coverslips in copper etchant for two hours. After rinsing the coverslips in distilled water, dry them with nitrogen, then gently buff the surface with lens paper to expose any posts that are still covered in cladding.
Soak the coverslips in copper etchant for another two hours, then rinse them again with distilled water and dry them with nitrogen. After surface passivation, zero mode waveguide coverslips can be paired with quartz slides using double-sided sticky tape to make microfluidic flow chambers for single molecule imaging. success of the colloidal template self-assembly depends critically on the relative humidity.
In a well-formed template, the region of beads is circular with borders defined by an opaque multilayered ring. In well-formed templates, most of the templates should contain hexagonally close-packed beads. There will be some defects between grains due to jamming during the evaporative sedimentation.
After copper deposition and dissolution of the polystyrene beads, copper posts should be below 150 nanometers in diameter. For a 300 nanometer copper deposition depth, posts are around 250 nanometers tall. After deposition of aluminum cladding and subsequent dissolution of the copper posts, waveguides should be visible by atomic force microscopy and be spaced about 550 nanometers apart.
Annealing the polystyrene templates for 20 seconds produced aluminum waveguides with a diameter of 118 nanometers, sufficiently small to cut off propagation of visible light. A typical field of ZMWs for imaging contains 3, 000 waveguides in a 40 by 80 micron field of view. Single molecule FRET was performed to test for ZMW functionality.
Single molecule FRET traces were detectable at all concentrations of ambient Cy5 fluorphore tested. In comparison, single molecules would only be detectable in TERF illumination with picomolar to low nanomolar concentrations. After zero mode waveguide fabrication, the devices can be passivated with polymers to reduce non-specific interactions during single molecule imaging.
The glass bottoms of the zero mode waveguides can also be etched slightly to enhance single molecule emission. Overall, this new zero mode waveguide fabrication method will allow researchers to more easily investigate dynamic biochemical processes, such as transcription or translation at concentrations and rates closer to those in the cell.
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Overview
This article describes a nanosphere lithography method for the parallel fabrication of zero mode waveguides, enabling single molecule imaging at low concentrations of fluorophores. The method utilizes colloidal crystal self-assembly to create a waveguide template on a glass coverslip.
Key Study Components
Area of Science
- Biophysics
- Nanotechnology
- Single molecule imaging
Background
- Zero mode waveguides are essential for studying single molecules.
- Traditional fabrication methods can be costly and require specialized tools.
- This method aims to simplify the process.
- Colloidal self-assembly is a promising technique for creating nanostructures.
Purpose of Study
- To present an accessible method for fabricating zero mode waveguides.
- To enable more researchers to conduct single molecule experiments.
- To facilitate studies at biologically relevant concentrations of fluorescent reagents.
Methods Used
- Use of optical borosilicate glass coverslips.
- Sonication in acetone for surface cleaning.
- Evaporative deposition of colloidal particles.
- Creation of a waveguide template for imaging.
Main Results
- The method is low-cost and does not require specialized tools.
- It produces effective waveguides for single molecule imaging.
- Researchers can achieve imaging at nano- to micromolar concentrations.
- The protocol is straightforward and reproducible.
Conclusions
- This nanosphere lithography method democratizes access to single molecule imaging techniques.
- It opens new avenues for research in single molecule biophysics.
- Future studies can leverage this method for various applications in biology.
Frequently Asked Questions
Executive Industry Relevance
This method enables single molecule imaging at micromolar fluorophore concentrations, overcoming a key limitation in biochemical assays where background fluorescence restricts usable concentrations to pico- to nanomolar ranges. By facilitating the study of molecular processes at cellular-relevant concentrations and rates, it supports more physiologically relevant target validation and mechanistic de-risking in early discovery. The accessibility of this low-cost fabrication approach expands the utility of zero mode waveguides beyond specialized core facilities to broader biopharma R&D laboratories.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of therapeutic hypotheses and pathway clarification by allowing single molecule observation at concentrations that reflect physiological ligand levels.
- Operational Value: Reduces reliance on expensive nanofabrication equipment, making single molecule enzymology accessible to more discovery teams.
- Predictive Confidence: Supports biological de-risking through direct visualization of single molecule interactions under near-native conditions.
Screening & Assay Development
- Scientific Value: Provides a platform for preparing validated biological systems that enable reliable compound evaluation at higher, more relevant fluorophore concentrations.
- Operational Value: Enhances assay standardization and reproducibility through parallel fabrication of thousands of zero mode waveguides using common lab equipment.
- Scalability: Supports platform reuse and scalability for downstream screening workflows by generating uniform waveguide arrays.
Translational & Preclinical Research
- Translational Continuity: Facilitates the study of dynamic biochemical processes such as transcription or translation at concentrations and rates closer to those in the cell, improving relevance to disease models.
- Mechanistic De-risking: Enables observation of single molecule FRET signals at micromolar concentrations, which are unattainable with traditional total internal reflection fluorescence (TERF) illumination.
- Risk-Adjusted Advancement: Supports data-driven decisions by providing quantitative, single molecule readouts that clarify mechanism and reduce ambiguity in lead optimization.
Pipeline & Workflow Integration
The method fits within the discovery continuum from early hypothesis testing through lead identification, enabling biophysical characterization that informs compound selection and optimization prior to preclinical evaluation.
- Discovery Biology: Supports hypothesis testing and biological de-risking by allowing direct observation of single molecule behavior in complex environments.
- Screening: Delivers assay-ready, reproducible nanostructures with quantitative outputs that enable comparison of ligand binding or enzymatic activity across conditions.
- Analytics: Provides single molecule fluorescence and FRET readouts that offer high-resolution kinetic and mechanistic insights beyond ensemble averages.
- Translational Research: Connects discovery findings to preclinical relevance by enabling studies at near-physiological concentrations and molecular crowding conditions.
- Enterprise Reuse: Establishes a reusable fabrication capability that can be shared across departments, reducing dependency on external nanofabrication services.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence in target validation by reducing mechanistic ambiguity through direct single molecule visualization.
- Operational Value: Improves standardization, reproducibility, and throughput via parallel fabrication using accessible equipment like thermal evaporators and hot plates.
- Strategic Value: Enhances capital efficiency and enables better go/no-go decisions by generating reliable biophysical data earlier in the discovery pipeline.
- Portfolio Impact: Supports risk-adjusted prioritization by providing mechanistic clarity that reduces late-stage attrition due to unexpected biological behavior.
Implementation Considerations
- Requires expertise in colloidal self-assembly, thermal evaporation, and surface cleaning protocols.
- Depends on access to a thermal evaporator, hot plate, sonicator, and basic chemistry lab equipment for metal deposition and etching.
- Necessitates standardization of humidity-controlled bead deposition to ensure consistent waveguide template formation.
- Involves adaptation considerations when applying the method to different substrate materials or waveguide metals (e.g., aluminum vs. gold).
- Includes practical limitations such as sensitivity to relative humidity during bead self-assembly and the need for careful handling to avoid template defects.
