Expansion Microscopy: High-Resolution Fluorescent Imaging with a Conventional Microscope

We are cell biologists investigating the role of microtubule and their associated function in different model organism. We aim to investigate their role in cellular organization, morphogenesis, ciliogenesis, and division. Over the past five years, we have contributed to the understanding of the circular molecular composition, notably through the use of expansion microscopy.

And by this, we reveal the key feature of their assembly and function. By combining expansion microscopy with cryofixation, our protocol enable high-resolution imaging while preserving the near-native structure of cellular element, which overcome the main limitation of super-resolution microscopy. So expansion microscopy, it's a low-cost and easy-to-implement techniques that give access to all the laboratory to super resolution.

To begin, use a syringe and a needle to fill each five-milliliter tube with one milliliter of extra dry acetone under a chemical hood. Place the tubes upright in a metal rack and put them in liquid nitrogen for freezing. Fill half of a 30-by-30 centimeters thick-walled polystyrene box with dry ice, and then fill the Vitrobot-type dewar with liquid nitrogen until evaporation stops.

Now, remove the spider from the plunging apparatus and fill the metallic plunging chamber with liquid ethane. Wait for 10 minutes to allow the ethane to reach equilibrium. Then, grab a 12-millimeter coverslip with thin tweezers and dab it on a tissue paper to soak up any excess medium from the coverslip.

Hold the coverslip halfway, using tweezers fitted with a clamping ring compatible with the cryo-plunger. Place the tweezers holding the coverslip into the cryo-plunger holder, and use Whatman paper to blot away any remaining medium. Activate the cryo-plunger to plunge the coverslip into the ethane solution contained in the metal chamber.

Then, quickly transfer the coverslip into the tube containing frozen acetone. Incubate the tubes containing the coverslips in dry ice at an angle of approximately 45 degrees. Place the closed container on an orbital shaker set at 50 to 100 RPM, and agitate overnight at four degrees Celsius to let the temperature rise gradually.

Next, remove most of the dry ice from the box. Continue agitation on the orbital shaker for an additional 45 minutes to let the temperature rise from minus 80 degrees Celsius to minus 20 degrees Celsius. Briefly open and close each tube to release internal pressure.

Then, transfer each coverslip into a 12-well plate or a suitable container pre-filled with pre-chilled 100%ethanol solution. After a five-minute incubation, rehydrate the coverslips through a graded ethanol series. After transferring the coverslips into PBS, place them under a microscope.

Use fine tweezers to gently scratch the surface and orient all coverslips with the correct side facing up. Place the coverslips into a four-well plate filled with 0.5 to one milliliter of 2%acrylamide and 1.4%formaldehyde solution in PBS. Then incubate the coverslips in this solution at 37 degrees Celsius without agitation.

Next, thaw 10%APS and the gelation solution on ice for 10 minutes before gelation. Prepare a humid chamber using a thin layer of wet tissue and Parafilm. Then store it at four degrees Celsius.

After 10 minutes, place the humid chamber on a cold block for use during gelation. Now, remove the coverslips from the protein anchoring solution and blot away excess liquid using tissue paper in two successive passes. Add APS to the gelation solution to reach a final concentration of 0.5%and vortex for two to three seconds.

Then, pipette two 35-microliter drops onto the Parafilm in the humid chamber and gently place each coverslip over a drop, with the cells facing downward into the gelation solution. Transfer the humid chamber to a 37-degrees-Celsius incubator for 30 to 60 minutes. For denaturation, use a biopsy punch tool with a 0.4 centimeter diameter to extract gel pieces, and place them into a six-well plate filled with one milliliter of denaturation buffer.

Agitate the plate for 10 to 15 minutes, until the gels detach from the coverslips, and transfer the detached gel pieces into 1.5 milliliter microcentrifuge tubes filled with fresh denaturation buffer. Incubate the gels for 90 minutes at 95 degrees Celsius. After incubation, transfer the gels into double distilled water for 10 minutes for washing.

Measure the diameter of the gels using millimeter paper to evaluate the gel expansion factor. Coat clean coverslips with approximately 200 microliters of polylysine solution. Incubate the coverslips for one hour at room temperature, then wash them three times with double distilled water to remove any excess polylysine.

After the coverslips are dry, store them at four degrees Celsius until further use. Place the gel properly orientated, with the cells facing up, onto lint-free paper and allow them to dry to eliminate excess water. Finally, mount the dried gels with the cells facing down onto Poly-D-Lysine-coated coverslips.

Cryofixation preserved the mitochondrial network in RPE1 cells more effectively than PFA fixation, as shown by NHS ester and ATP5A staining, and allowed resolution of mitochondrial cristae, which were not visible with PFA fixation. Cryofixation resulted in better preservation of dynamic microtubules, including cytoplasmic and astral microtubules in mitotic RPE1 cells, compared to PFA fixation. Trypanosoma brucei, cryofixation better preserved the architecture of the mitochondrion and general cellular structure compared to PFA fixation, as visualized with TDH and NHS ester staining.

Additionally, cryofixation preserved the endoplasmic reticulum better than PFA fixation based on BIP staining. Cracks were observed in cryofixed RPE1 cells, but these did not disrupt the ultra structure of organelles, such as mitochondria or microtubules. Poor cryofixation was indicated by bubble-like structures and wavy microtubules, accompanied by loss of membranous organelle integrity.

Expansion quality was dependent on the clarity of sodium acrylate solutions, with usable solutions appearing colorless or slightly yellow and translucent, while unusable ones appeared cloudy and orange.