High-Throughput Total Internal Reflection Fluorescence and Direct Stochastic Optical Reconstruction Microscopy Using a Photonic Chip
November 16th, 2019
Chip-based super-resolution optical microscopy is a novel approach to fluorescence microscopy and offers advantages in cost effectiveness and throughput. Here, the protocols for chip preparation and imaging are shown for TIRF microscopy and localization-based super-resolution microscopy.
The main goal of this manuscript is to present an imaging procedure of newly-developed photonic chip-based TIRF microscopy and single-molecule localisation microscopy, such as dSTORM. The main component here is the photonic chip that are made of series of optical waveguide. The light inside the optical waveguide is guided based on total internal reflection.
Some of the light is present on the surface in the form of the evanescent waves. Those evanescent waves, they decay exponentially away from the surface, with a penetration depth of a couple of hundred nanometers. This makes them suitable for TIRF illumination.
TIRF illumination via the optical waveguide is available over an extremely large area, which is defined by the waveguide geometry, and is thus ideally suited for high-throughput imaging. The key difference between a traditional TIRF and dSTORM setup as compared to our approach is that we use a photonic chip to illuminate the sample, instead of sending light through an imaging objective lens. Our approach decouples illumination and the collection light part, opening new imaging possibilities.
Place the chip in the glass Petri dish using a wafer tweezer, and cover completely with a 1%Hellmanex solution. Place the Petri dish on a hotplate at 70 degrees for 10 minutes. While still on the hotplate, rub the surface with a cleanroom tissue swab.
Remove the chip from the Petri dish. Rinse with at least 100 milliliters of deioned water. Rinse with at least 100 milliliters of isopropanol, taking care that the solvent does not dry on the surface to prevent evaporation stains.
Rinse with at least 100 milliliters of deioned water. Blow the chip dry with a nitrogen gun. Prepare a later of 150 micrometers PDMS by spinning it in a Petri dish.
Use a scalpel to cut an approximately 1.5 by 1.5 centimeter frame from the PDMS layer. Lift the frame from the Petri dish with a tweezer. Deposit it flat on a clean and polished chip.
The sample is now ready for cell seeding. After cell seeding, remove the chip from the media. Use a pipet to remove any excess fluid from outside the PDMS chamber.
Remove the current fluid from inside the PDMS chamber with a pipet while adding approximately 60 microliters clean PBS at the same time. Replace the PBS with 60 microliters clean PBS, and let it incubate for one minute. Repeat the previous step, letting it incubate for five minutes this time.
Remove the PBS, and replace it with 60 microliters of the dye solution. Leave the sample to incubate for around 15 minutes, shielding it from light. Wash the sample with PBS by using the previously-described procedure.
Remove the PBS, and replace it with 40 microliters of the imaging buffer at the same time. Place a coverslip on top, preventing air bubbles from forming underneath. Gently press the coverslip against the imaging chamber to remove any excess media.
Use a pipet to remove any excess media outside the coverslip. Clean the area outside the coverslip with a water-moist swab to avoid crystals formed by dried immersion media residues. This version of the setup consists of three main components.
The microscope with the filter holder, white light source, camera, and objective revolver. The coupling stage, which is a three-axis piezo stage, with a fiber-coupled laser, and a coupling lens. The sample stage, which is a one-axis manual stage, with tip and tilt, with a vacuum holder.
Both the coupling and sample stage are mounted on a two-axis motorized stage for sample translation. Place the chip on the vacuum truck, with the coupling facet towards the coupling objective. Make sure the chip is approximately one focal length away from the coupling objective.
Turn on the vacuum pump. Turn on the laser to one milliwatt. Roughly adjust the chip height so that the beam hits the edge of it.
Turn off the laser. Turn on the white light source. Choose a low-magnification objective lens, for example, a 10x.
Focus the microscope on a waveguide. Translate the microscope along the waveguide to see if it is well aligned with the optical path. Move the microscope to the coupling edge.
Turn on the laser at one milliwatt or less. Translate the microscope along the coupling edge to find the laser light. Focus the beam on the chip edge.
Adjust the coupling stage along the optical path in the direction that reduces the laser beam spot size until it disappears. The beam is now either above or below the chip surface. Adjust the coupling stage height until the beam spot reappears and is maximized.
Repeat the two previous steps until the laser forms a focused spot. Move the focused spot to the waveguide of interest. Translate the microscope a short distance away from the edge so that the focused beam spot is no longer visible.
Turn off the white light. Adjust the contrast. If the waveguide is guiding, the scattered light along the waveguide should be clearly visible.
Adjust the axis of the coupling stage to maximize the scattered light intensity. Turn off the laser. Turn on the white light.
Adjust the contrast if necessary. Navigate to the imaging region. Focus with the desired imagine objective.
Turn the white light off. Insert the fluorescence filter, and turn the laser power to one milliwatt. Set the camera exposure time to approximately 100 milliseconds.
Adjust the contrast as needed. Ensure that the coupling is still optimized. Locate a region of interest for imaging.
Turn on the piezo stage looping to average out nodes. Capture at least 300 images. Load the captured image stack to Fiji using a virtual stack.
From the image menu in Fiji, choose Stacks, and Z Project. Calculate the TIRF image by choosing Projection type, Average Intensity. Turn on the laser to one milliwatt, and set the camera exposure time to 30 milliseconds.
Adjust the contrast and focus. Increase the laser power until blinking is observed. Zoom in on a small region of the sample.
Adjust the contrast. Capture a few images to see if the blinks are well separated. Adjust the camera exposure time for optimal blinking.
Turn on the piezo stage looping. Record an image stack of at least 30, 000 frames, depending on the blinking density. Open Fiji, and load the dSTORM stack as virtual images.
Adjust the contrast if necessary. Use the rectangle tool to select the area you want to reconstruct. Open Run analysis in the ThunderSTORM plugin in Fiji.
Set the basic camera settings in ThunderSTORM, corresponding to your device. Remaining default parameters are usually satisfactory. Start the reconstruction.
The localization list provided by the reconstruction software is filtered to remove unspecific localisations. An additional drift correction is applied if necessary. The main difference between chip-based imaging and traditional imaging is in the instrumentation and data acquisition.
The quality of the reconstructed images can therefore be assessed in the same manner as an image from a commercial super-resolution microscope. Since multi-mode waveguides are used, the resulting image might, however, exhibit inhomogeneous excitation if too few images are collected. This is shown in panel a.
Increasing the amount of excitation patterns should result in inhomogeneous excitation, as shown in panel b. Here we have imaged liver sinusoidal endothelial cell, with fluorescently-labeled plasma membrane. Panels a and b are diffraction-limited images.
Panel c shows a diffraction-limited image of the inset in panel b. Panel d shows a dSTORM image of the same region. Liver sinusoidal endothelial cells have nano-sized fenestrations in their plasma membrane, which are clearly visible in the super-resolution image in panel d.
One of the main advantages of chip-based super-resolution imaging is the large field of view that is achievable. Panel a shows a 500 micron by 500 micron-large dSTORM image of liver sinusoidal endothelial cells with fluorescently-labeled microtubulin. Panel b shows the magenta inset from panel a, with both diffraction-limited and super-resolution images for comparison.
Panel c shows the green inset from panel a. The resolution of the captured image is 77 nanometers. In this video, we have performed large field of view TIRF and dSTORM imaging of liver sinusoidal endothelial cells using a photonic chip for illumination.
Our method is less complex, more compact, and more flexible than the conventional way of performing TIRF using a microscope objective of a predetermined numerical aperture and low field of view. Localisation microscopy, such as dSTORM, is one of several super-resolution imaging technique that we have explored using photonic chip. For example, light can be much more tightly confined inside a high-refractive index optical waveguide material than it is possible using an imaging objective lens.
This property of chip illumination has found application in increasing the resolution of intensity-fluctuation-based optical microscopy method and structured illumination microscopy. In addition, photonic chips also benefits from miniaturization, cost effectiveness, and a simple optical setup. Being an integrated technology makes it compatible with other on-chip optical functions.
Altogether, this makes it possible for retrofitting into conventional diffraction-limited microscopes, allowing for super-resolution at a low expense.
Overview
This article presents a novel chip-based super-resolution optical microscopy technique, focusing on TIRF microscopy and single-molecule localization microscopy. The protocols for chip preparation and imaging are detailed, highlighting the advantages of this approach in cost-effectiveness and throughput.
Key Study Components
Area of Science
- Fluorescence microscopy
- Super-resolution imaging
- Photonic chip technology
Background
- Traditional TIRF microscopy relies on objective lens illumination.
- Photonic chips utilize optical waveguides for light guidance.
- Evanescent waves enable high-resolution imaging at the surface level.
- This method enhances imaging area and throughput.
Purpose of Study
- To demonstrate a new imaging procedure using photonic chip-based TIRF microscopy.
- To compare traditional methods with the chip-based approach.
- To provide detailed protocols for chip preparation and imaging.
Methods Used
- Preparation of photonic chips using optical waveguides.
- Cell seeding and incubation protocols for sample preparation.
- Imaging setup involving a microscope, laser, and camera.
- Image capture and processing using Fiji software.
Main Results
- Successful demonstration of high-throughput imaging capabilities.
- Clear visibility of scattered light along the waveguide.
- Effective localization of single molecules using the new setup.
- Improved imaging resolution compared to traditional methods.
Conclusions
- The chip-based approach offers significant advantages in fluorescence microscopy.
- Decoupling illumination and collection enhances imaging possibilities.
- This method paves the way for future advancements in super-resolution microscopy.
Frequently Asked Questions
Executive Industry Relevance
Chip-based nanoscopy decouples excitation and collection pathways, enabling large field-of-view TIRF imaging with low magnification optics. This approach supports high-throughput super-resolution imaging while maintaining resolution comparable to traditional methods, addressing discovery-stage bottlenecks in target validation and phenotypic screening. The technique enhances predictive confidence by providing quantitative, reproducible subcellular imaging data across scalable sample areas.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of subcellular structures like liver sinusoidal endothelial cell fenestrations and plasma membrane organization at 76 nm resolution.
- Operational Value: Decoupled illumination and collection allow use of low magnification lenses, increasing field of view for population-level target engagement analysis.
- Predictive Value: Large-area dSTORM imaging supports statistical robustness in target localization and heterogeneity assessment.
Screening & Assay Development
- Scientific Value: Uniform evanescent wave excitation across waveguide-defined areas enables consistent single-molecule localization for assay readouts.
- Operational Value: Compatibility with standard microscopes and on-chip integration supports assay standardization and reuse across discovery campaigns.
- Scalability: Demonstrated 500 µm x 500 µm imaging area enables multiplexed condition testing in a single acquisition.
Translational & Preclinical Research
- Translational Continuity: Disease-relevant LSEC imaging validates applicability to endothelial pathophysiology models.
- Mechanistic De-risking: Super-resolution visualization of nano-scale membrane features aids in understanding drug-target spatial relationships.
- Preclinical Alignment: Resolution and throughput support biomarker localization studies in primary or stem-cell-derived systems.
Pipeline & Workflow Integration
The method fits within early discovery to lead identification workflows, providing super-resolution readouts that inform target confidence and assay reproducibility prior to compound screening.
- Discovery Biology: Supports hypothesis testing via direct visualization of target localization and clustering in native membrane environments.
- Screening: Enables assay readiness through quantifiable, high-density localization data from large sample regions.
- Analytics: Generates filtered localization lists and drift-corrected reconstructions for objective comparison of experimental conditions.
- Translational Research: Connects to preclinical validation through imaging of disease-relevant cellular ultrastructure in primary cell models.
- Enterprise Reuse: Photonic chip infrastructure can be retained across projects, reducing retooling costs for super-resolution capable imaging.
Operational & Enterprise Impact
- Scientific Value: Predictive confidence in target validation through nanoscale resolution of membrane protein organization and dynamics.
- Operational Value: Standardized chip preparation and imaging protocol ensures reproducibility across users and sites.
- Strategic Value: Enables go/no-go decisions based on subcellular target distribution data, reducing late-stage attrition from mechanistic misunderstanding.
- Portfolio Impact: Risk-adjusted prioritization of targets supported by super-resolution spatial evidence in relevant cellular contexts.
Implementation Considerations
- Expertise in photonic chip handling, fluorescence microscopy, and single-molecule localization analysis required.
- Instrumentation includes laser source, piezo stage, vacuum holder, and fluorescence filter set compatible with TIRF and dSTORM.
- Cross-team standardization needs include chip cleaning procedures, PDMS chamber fabrication, and Fiji/ThunderSTORM analysis pipelines.
- Adaptation considerations involve optimizing excitation patterns for homogeneous illumination across different waveguide geometries and sample types.
- Practical limitations include potential inhomogeneous excitation with insufficient pattern averaging and minor resolution trade-off versus high-NA objective TIRF.
