Figure 1. High-throughput expansion microscopy (HiExM) enables gel formation and expansion in a 96-well cell culture plate.
(a) Schematic representation of HiExM devices showing the key features highlighted in color. (b) Example devices used in 96-well cell culture plates. (c) Brightfield image of the conical post-tip shows the pattern of grooves that mediate fluid retention. (d) Fluid retention at the conical post-tip of the device. Silhouettes taken by an optical comparator of the profile of a single post suspended above a surface (left) and in contact with a surface (right) show a fluid droplet interacting with the device. Upon device insertion, the gel solution fills the space under the conical post tip, forming the toroid gel. (e–h) Schematic of HiExM gel deposition and expansion workflow. (e) The device is immersed in a shallow reservoir of gel solution. (f) Upon removal, the tip of each device post retains a small volume of gel solution. (g) Gel solution is deposited by the device into the well centers of the cell culture plate. Brightfield image (right) shows gel geometry and size prior to expansion. Note that gels deposited in HiExM cover ~1.1 mm2 of the well surface to accommodate the expanded gel, and do not include cells outside the gel footprint. (h) Polymerization and expansion are performed with the device in place. Brightfield image (right) shows gel geometry and size after expansion.
Figure 1—figure supplement 1. Schematic depiction of high-throughput expansion microscopy (HiExM) device with displayed features.
The device was designed with three different post lengths where the four center posts are the longest, the two pairs of posts on the ends near the pressure struts are the shortest, and the middle posts are an intermediate length. When the device is inserted into the well plate, the center four posts contact the culture surface first. As downward pressure is applied to the ends of the device (by the user pressing down above the pressure struts), the spine of the device bends at the notches such that all posts contact the plate. These design features allow for the reproducible deposition of gel solution droplets and the reproducible formation of toroidal gels within the well constraints.
Figure 1—figure supplement 2. Overall schematic of high-throughput expansion microscopy (HiExM) comparing standard chemistry and photoinitiation.
(A) For standard expansion microscopy (ExM), the two initiating components (ammonium persulfate, APS and tetramethylethylenediamine, TEMED) were added to cells in wells in a two-step process to control the polymerization rate. In the first step, a droplet of TEMED-containing gel solution is deposited in the well and the device is removed. In the second step, a second identical device delivers a droplet of APS-containing gel solution to the same well with the TEMED-containing droplet to initiate polymerization. The well plate is also kept on ice for 15 min while the APS and TEMED are mixed, followed by heating the well plate at 50 °C for 5 min to initiate rapid polymerization. Additionally, the protocol is performed in a glove bag purged twice with nitrogen. (B) The use of photoinitiation HiExM as described in Methods allows for one-step polymerization. The Irgacure 2959 gel solution is deposited and polymerization is initiated by irradiation with a UVA metal halide lamp.
Figure 1—figure supplement 3. Detailed workflow of the high-throughput expansion microscopy (HiExM) protocol using photoinitiation.
HiExM can be used with cells cultured in commercially available 96-well plates. After conducting an experiment, cells can be fixed and stained as normal, followed by the addition of the anchor molecule Acryloyl-X. The following day, Acyloyl-X is aspirated, washed once with PBS and once with DI water, and then aspirated again to leave cells dry. It is important to ensure that no residual liquid remains in the well. Following aspiration, the device is used to deposit gel droplets on cells. Deposited gels are left for ~90 s followed by a 70- s exposure to UV light to initiate polymerization. Proteinase K solution is then pipetted into the wells and after 6 hr, the plate is submerged in ~4 L of water in a beaker and left suspended over a stir bar overnight. Water is then carefully aspirated from the wells and a layer of mineral oil is applied to each well to prevent evaporation and to stabilize the gel position for imaging. All steps in this procedure are performed at room temperature.
Figure 1—figure supplement 4. CF conjugated antibodies yield robust signal and resistance to photobleaching in Irgacure high-throughput expansion Microscopy microscopy (HiExM).
Representative images of A549 cells stained with α-Tubulin antibodies and secondary antibodies conjugated to CF dyes at different wavelengths before (left) and after (right) exposure to UV light as described in the HiExM protocol. Stained cells were exposed to UV light in the presence of Irgacure 2959 in 0.1% PBS. Histograms representing the raw pixel values from these images are shown on the right, where the number on each plot represents the peak pixel value for that image. Decreased peak pixel values indicate a loss of signal due to photobleaching.
Figure 1—figure supplement 5. Acryloyl-X (AcX) and ProteinaseK titration.
Signal-to-noise ratio was determined by measuring the average fluorescence intensity in an area of the cell and an area of the background for α-tubulin stained A549 cells from images obtained using the Opera Phenix. Optimum signal-to-noise was found at concentrations of 50 µg/mL AcX and 1 U/mL ProteinaseK.
Figure 1—figure supplement 6. Residual hoechst signal occasionally remains underneath high-throughput expansion microscopy (HiExM) samples.
The lowest image planes (top row) of three example images that show residual hoechst signal. Higher planes from the same images (bottom row) show nuclei within the sample.
Figure 1—figure supplement 7. Schematic depiction of the 24-well plate high-throughput expansion microscopy (HiExM) device.
This device was designed to form one gel in a well of a standard 24-well cell culture plate and manufactured with injection molding (Protolabs).
Figure 1—animation 1. Example of expansion process in high-throughput expansion microscopy (HiExM) with ammonium persulfate (APS)/tetramethylethylenediamine(TEMED) chemistry.
Tubulin-stained A549s were monitored after gel formation with digestion buffer on. This video, taken in real time, shows how cells treated with this protocol tend to ‘stick’ to the cell culture substrate as the gel expands. Cells are torn off the culture substrate by the expanding gel, often leaving behind residual cellular material stuck to the culture substrate.
elife-96025-fig1-video1.gif (10MB, gif)
Figure 1—animation 2. Example of expansion process in high-throughput expansion microscopy (HiExM) with Irgacure chemistry.
Tubulin-stained A549s were monitored after gel formation with digestion buffer on. This video, taken at 10X speed, shows cells expanding normally in HiExM in the presence of the digestion buffer. The post of the device is close to the bottom-left of the image.
elife-96025-fig1-video2.gif (18.2MB, gif)
