Stochastic Micro-Pattern for Automated Correlative Fluorescence - Scanning Electron Microscopy

Studies of cellular surface features gain from correlative approaches, where live cell information acquired by fluorescence light microscopy is complemented by ultrastructural information from scanning electron micrographs. Current approaches to spatially align fluorescence images with scanning electron micrographs are technically challenging and often cost or time-intensive. Relying exclusively on open-source software and equipment available in a standard lab, we have developed a method for rapid, software-assisted alignment of fluorescence images with the corresponding scanning electron micrographs via a stochastic gold micro-pattern. Here, we provide detailed instructions for micro-pattern production and image processing, troubleshooting for critical intermediate steps, and examples of membrane ultra-structures aligned with the fluorescence signal of proteins enriched at such sites. Together, the presented method for correlative fluorescence – scanning electron microscopy is versatile, robust and easily integrated into existing workflows, permitting image alignment with accuracy comparable to existing approaches with negligible investment of time or capital.


patterns.
To depict the variability of gold micro-pattern in size, a 100x zoom into the global map of a 18 mm glass coverslip coated with gold micro-pattern is shown. Note that the final frame (180 μm x 180 μm) is smaller than a single image acquired with a sCMOS camera on a 60x objective (210 μm x 210 μm).

Supplementary Movie 2: Rotational alignment of micro-pattern from light and electron microscopic images via ALIGN macro.
To identify the optimal angle for image alignment, the micro-pattern from SEM back-scatter image (red) is rotated relative to the micro-pattern from the brightfield image (green). Alignment score for the relative angle is shown below.

Micro-Pattern productiontime requirements and troubleshooting
All procedures for the production of gold micro-pattern were optimized for the application in correlative light-electron microscopy (Supplementary Figure 9). A detailed description on critical points for all steps can be found below.
In order to achieve uniform layer thickness and secure good binding of gold to the glass (step a), 5 minute long washing steps in acetone and ethanol are recommended.
For coating the coverslip (step b), any type of gold deposition can be utilized. We have tested thermal vacuum deposition and sputter-coating, and prefer the latter as it is faster. Compared to deposition by evaporation, the sputter-coated structure is supposedly coarser and slightly less homogeneous.
However, we did not observe visible differences in the quality of the micropattern. In our hands, preparation of an approximately 25 -30 nm thick gold layer took about 15 minutes.
For creating the later micro-pattern on the gold-coated glass coverslip (step c), positive photo-resist was sprayed under a shallow angle (i.e. from the side) at a distance of ~20 -30 cm. To reduce the size-distribution of the micropattern, droplet dispersion can be changed using either a finer nozzle or smallmeshed grids. By changing the angle, the distance or the dispersing tool, dot size range and dot density can be controlled. Note that photo-resist droplets S13 tend to fuse, thus creating a homogenous layer. By repeating spraying/drying cycles (~ 30 s) with small amounts of photo-resist, it is possible to create pattern with distinct dot sizes and complex shapes. Please avoid exposure to direct sunlight, as photo-resist is light sensitive.
To create gold micro-pattern (steps d,e), the sprayed photo-resist first has to dry onto the gold. This can be done either by drying at room temperature overnight, by soft-baking at low temperature in a heating chamber for 1-2 hours, or with a hard bake for ~10 minutes at high temperature (note: times and temperature vary between different photo-resists. See instructions of your product for details). We tested all three possibilities. While all worked, we observed that the type of drying influences its attachment strength of photo-resist to the gold layer, which is critical for the following washing procedures to remove the photo-resist (see step f below). For etching the uncovered gold areas, a diluted version of aqua regia was used. With increasing micro-pattern density, it is recommended to increase the amount of water in the acidic solution to prevent under-etching Note, a duration of 15 -20 seconds should not be exceeded.
Afterwards, it is important to immediately stop the etching process by washing off the acid. We find that washing in water can cause gold lift-off. In contrast, dipping the coverslip into plating medium (also to be used later on for plating cells) allowed to wash off the acid with no noticeable gold loss. After a few seconds, the etched micro-pattern coverslip was transferred into a small beaker containing ethanol and dried. S14 Finally, the photo-resist was removed (step f) by leaving the coverslips in DMSO for ~ 2 minutes. DMSO was then washed off with ethanol and let dry (~20 s). Note that left-over photo-resist affects the quality of the SEM image, but not cell growth or cell viability. To ensure that all photo-resist had been removed by DMSO, coverslips can be inspected under a light microscope: Clean micropattern appear as shiny particles with gold reflection, whereas micro-patterns with residual photoresist appear rainbow/greenish. As DMSO will leave streaks on the coverslip surface, repeated washing steps may be required. The micropatterned coverslips can then be stored in a dust-free environment until use.
Before plating, the coverslips are cleaned again with ethanol, dried and put for sterilization 10 minutes under UV-light. Generally, the micro-pattern should not get into contact with pure water as this may cause lift-off of gold dots.
Further, care should be taken in terms of scratching the surface with forceps during handling (an example can be found in Supplementary Figure 4c, panel g). During plating and imaging no additional care is necessary. S15