Photonic-chip assisted correlative light and electron microscopy

Correlative light and electron microscopy (CLEM) unifies the versatility of light microscopy (LM) with the high resolution of electron microscopy (EM), allowing one to zoom into the complex organization of cells. Here, we introduce photonic chip assisted CLEM, enabling multi-modal total internal reflection fluorescence (TIRF) microscopy over large field of view and high precision localization of the target area of interest within EM. The photonic chips are used as a substrate to hold, to illuminate and to provide landmarking of the sample through specially designed grid-like numbering systems. Using this approach, we demonstrate its applicability for tracking the area of interest, imaging the three-dimensional (3D) structural organization of nano-sized morphological features on liver sinusoidal endothelial cells such as fenestrations (trans-cytoplasmic nanopores), and correlating specific endo-lysosomal compartments with its cargo protein upon endocytosis.

: Scheme of fabrication steps of the waveguide chips. A) 1 mm thick silicon wafer. B) Approximately 2 µm thick SiO2 created through thermal oxidation. C) Deposition of 150 nm thick Si3N4. D) Photolithographic patterning followed by etching of Si3N4. Waveguide widths for imaging range between 25 to 500 µm. E) Deposition and patterning of absorbing layer consisting of 200 nm thick SiO2 (bottom) and 100 nm thick polycrystalline silicon (on top). F) Deposition and patterning of 1.5 µm thick SiO2 cladding for imaging regions and landmarks.

Supplementary Note 2 "Landmark visibility"
Landmarks were used as a coordinate system in optical ( Figure S2 A-C) and electron microscopy ( Figure  S2 D-E). Figure S2 A-C shows the high contrast of landmarks under brightfield illumination at the full field of view of 4x, 20x, and 60x magnification, respectively. High contrast and thus visibility allow for easy position readout. Figure S2 D-E are scanning electron microscopy images of different chips after the resin step, showing resin thickness variations that can occur after centrifugation and polymerization. A prerequisite for optimal resin thickness, thus landmarks visibility, was to centrifuge the chips in vertical position at 37°C for 30 min. Despite different degrees of visibility, the landmarks can still be identified in the scanning electron microscope.

Supplementary Note 3 "Large FOV and multiplexing"
Making use of a waveguide chip as a substrate and illumination source decouples the typical dependency between excitation and collection optics. It offers the generation of evanescent field without the necessity of a specialized high magnification/numerical aperture TIRF lens, enabling free choice of imaging objective. This property is of advantage for large field of view TIRF-imaging applications. Here, an isolated LSEC was identified and selected by only using the signal from the membrane stain emission channel under 4x magnification, after which a 60x multi channel image was taken (Fig. S3). For conventional, objective-based TIRF setups, multicolor imaging is cumbersome as it requires repositioning of the beam to fulfill the total internal reflection condition when changing between excitation wavelengths. The waveguide setup allows for easy multiplexing, as an achromatic coupling lens will provide the same beam spot for coupling between different laser lines without the necessity of mechanical adjustments. As propagation losses in the waveguide are higher for shorter wavelengths, compensation in the illumination intensity might be necessary. It was however observed that the intensity of the fluorophore stain had at least a similar influence in the signal to noise ratio.

Supplementary Note 4 "Waveguide setup"
A scheme of the setup is shown in Figure S4 A) Laser beams at three conventional bioimaging wavelengths (561 nm 500 mW and 660 nm 500 mW-Cobolt, 488 nm 200 mW -Oxius) are expanded to slightly overfill the back aperture of the coupling objective (Olympus, 50x / 0.5 NA). The laser beams are aligned using dichroic mirrors (Edmund Optics) towards the coupling objective mounted on a 3 axis flexure stage featuring piezo motors (Thorlabs Nanomax). The waveguide chip is mounted on a separate 3 axis flexure stage (Thorlabs Nanomax) with a vacuum chuck (Thorlabs) to hold the chip. A modular microscope setup (Olympus BXFM) mounted on motorized translation stages for two axis translation (Thorlabs) provides brightfield illumination and acquires images at different magnifications (4x, 20x / 0.45 NA, 60x / 1.2 NA water immersion, all Olympus). High magnification is used with a z-piezo system (PIFOC, PI) for fine adjustment. A homemade adapter holds a cage filter wheel (Thorlabs) between the microscope body and the tube lens (1x). For each excitation wavelength, a long pass and a band pass filter were used (488 LP and 520 ± 36 nm, 561 LP and 591 ± 43 nm, 664 LP and 692 ± 40 nm, AHF Analysentechnik), and the images recorded with a sCMOS camera (Hamamatsu Orca Flash). Figure S4: Waveguide setup A) Experimental-set-up for waveguide-based optical micro-/nanoscopy. B) Waveguide chip with mounted PDMS frame and coverslip sealing the imaging region. C) Single-mode fiber pigtailed to waveguide facet as an alternative objective-based coupling method, which could be useful for integrated CLEM.

Supplementary Note 5 "Homogeneous illumination with multimoded waveguides"
Recently, for Si3N4 waveguide platform operating at visible wavelengths, a single condition carrying only the fundamental mode was demonstrated using a rib waveguide geometry 3 for 150 nm thick and 1.5 µm wide Si3N4 waveguides. Adiabatic tapering can increase the width of the structures for imaging, but required taper length for widths of hundreds of micrometers would be of several millimetres 1 . In this work, we employed a stripe geometry fully etching the waveguide surrounding. For the width of the waveguide used in this work (25 µm), the waveguide supports several modes 2 . The presence of multiple modes creates interference patterns and so uneven illumination on the surface. This can be reduced by scanning the coupling beam over the waveguide width ( Figure S5) and averaging the mode beating pattern generating a uniform image 4 . This allows for the use of waveguide structures of several hundreds of micrometers in width. In Figures 2d and 4a, striations from the mode beating pattern are still visible. Those can be attributed to the relatively narrow width of the waveguide (25 µm), but also to the limited travel range of the piezo motor (20 µm) utilized to scan the input coupling. Wider waveguides, meaning more modes, and a stage with longer travel range will ideally provide striation free images, as it can be seen in previous publications 1, 3,4 . Alternatively, a galvo mirror can deflect the beam on the back aperture of the coupling objective, scanning the waveguide input at high frequency. This will require only a single or a small number of images compared to the translation stage used in this work, where typically 100 images were acquired for averaging. However, as it is difficult to tune the galvo mirror for an ideal deflection angle, it comes at cost of lower laser intensities reaching the waveguide. Figure S5: Left: Multimode waveguides provide uneven illumination through interference patterns between modes. These patterns can be varied when moving the coupling beam along the input facet. Right: Averaging images of these mode variations results provide an almost homogeneous illumination pattern.

Suppplementary Note 6 "Evanescent field penetration depth"
Simulations were performed to visualize the decay of the evanescent field above the waveguide. The geometry consisted of a 150 nm thick silicon nitride structure with different widths lying on a silicon dioxide substrate and surrounded by water. The software Fimmwave was used to calculate the effective refractive index of the fundamental TE and TM modes, as well the highest TE mode found for a given width (Figure S6 A). With the real part of the effective refractive index, an analytical solution 5 was used to determine the evanescent field decay ( Figures S6 A and B). It can be seen in the table of Figure S6 A that the penetration depth, expressed through the decay to 1/e (ca. 37%) of the initial evanescent field intensity, is higher for longer wavelengths and TM polarization. The intensity difference between the polarization states is also higher at longer wavelengths (ca. 30%), and could be explored to modulate the penetration depth through rotation of the polarization of the incoupling beam. When it comes to the span of modes present for, e.g., pure TE polarization, it can be seen that the mode averaging for homogeneous illumination practically does not affect the penetration depth with differences below 1 nm. This negligible difference between the modes is also kept for different waveguide widths. Figure S6: Simulations for the evanescent field decay in a 150 nm tall silicon nitride waveguide. A: penetration depth for different widths, polarization and wavelengths. B: Evanescent field decay for fundamental modes at two different wavelengths and polarization states.

Supplementary Note 7 "Work flowchart"
The flowchart in Figure S7 provides an overview of the processing steps from chip preparation, cell seeding and staining, light microscopy imaging, sample preparation for electron microscopy and FIB-SEM imaging. Figure S7: Work flowchart.

Supplementary Note 8 "Waveguide imaging resolution"
To benchmark the resolution for the chip-based TIRF images, fluorescent beads with a diameter of 200 nm (TetraSpeck microspheres, Invitrogen) were deposited on a chip surface, excited at 660 nm and imaged with the 60x/1.2 NA water immersion objective used for the cell experiments. Figure S8 A shows a section of an acquired fluorescence image, with S8 B demonstrating how three of the indicated line profiles were fitted with a Gaussian curve to estimate the resolution through the fit's full width at half maximum (FWHM). The six points marked in S8A provide an averaged resolution of 390 nm with a standard deviation of 15 nm, the theoretical diffraction-limited resolution for the used objective at the emission wavelength of ca. 680 nm being 346 nm. For the achievable resolution in the dSTORM experiments, a monomolecular layer of fluorophore was used to determine the localization precision of the blinking sites. For this end, 100 µl of a PBS solution containing 100 nm streptavidin conjugated Alexa Fluor 647 (Invitrogen) was mixed with 100 µm 0.1% poly-L-lysine solution. After incubating at the chip surface for 20 minutes, three wash steps with distilled water remove the excess dye on the chip. Buffer media is added, and the sample is ready for measurements after placing a coverslip on top. Figure S8C presents the frequency of the different dimensions localized with the algorithm of the ThunderSTORM plugin for Image J 6 , indicating a potential localization of 9.4 nm or a resolution of 22.1 nm at FWHM for the 60x/1.2 NA objective.