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.
