This protocol details the method of ultrastructure expansion microscopy in three in vitro lifecycle stages of Trypanosoma cruzi, the pathogen responsible for Chagas disease. This protocol offers an alternative to the current super resolution imaging and electron microscopy techniques, with the advantage of being compatible with conventional microscopes found in most biology labs and in machine core facilities. It uses common techniques for most research labs to obtain images that enables three-dimensional reconstruction.
The protocol facilitates the study of nanoscale structures in trypanosomatids allowing the inspection of a population of cells and imaging the entire volume of a cell of interest at a high resolution. It is particularly useful for science-specific cell types in transient phases of the cell cycle and differentiation. To gain even more resolution, we will use the combination of ExM with super resolution microscopic techniques in order to reach resolutions below 20 nanometers.
Begin by preparing Trypanosoma cruzi epimastigote suspension from a log phase culture in LIT medium supplemented with 10%FCS. Centrifuge the suspension at 5, 000 G for 10 minutes at room temperature. Wash the pellet two times with PBS, then resuspend in 200 microliters of PBS.
Adhere the suspension to a round 12 millimeter cover slip coated with Poly-D-Lysine and incubate it at room temperature for 15 to 20 minutes before cross-linking prevention. Collect the supernatant of a Vero cell monolayer infected with the trypomastigotes four days post-infection. Using a Neubauer cell counting chamber, determine the concentration of the trypomastigotes.
Centrifuge the cell suspension at 7, 000 G for 10 minutes at room temperature. After rinsing and resuspending the cells in PBS, transfer the cell suspension to a lysine-coated cover slip and incubate the cover slip at room temperature for 10 to 15 minutes before cross-link prevention. To obtain amastigotes from infected Vero cells, lay a sterile 12 millimeter round cover slip at the bottom of a 24 well tissue culture plate.
Next, prepare a suspension of Vero cells in DMEM supplemented with 2%FCS, and seed 500 microliters of the suspension into each well. Incubate the plate overnight to ensure cell attachment. Then, wash the cells twice with 500 microliters of sterile PBS.
Add T.cruzi trypomastigotes into the cells and incubate as earlier for six hours. Start by adding 0.5 milliliters of cross-linking prevention solution to each well of a 24-well plate. Then, submerge the 12 millimeter Poly-D-Lysine-coated cover slips with adherent parasites.
After filling the empty wells with water to reduce evaporation, seal the plate with a sealing film and incubate. To gellate the samples, first assemble a humid chamber in a Petri dish by placing a sealing film on top of tissue paper. Then, wet the tissue paper with water and incubate at minus 20 degrees Celsius for 20 minutes to cool it.
Lay the cool humid chamber on ice. Next, using a three milliliter Pasteur pipette, aspirate the cross-linking prevention solution from the wells of the 24 well plate. Now, use tweezers to remove the cover slips and place them on the tissue paper with the parasites facing up.
Take 90 microliters of monomeric solution in a tube and add five microliters each of thawed TEMED and APS. Vortex the mixture for two to three seconds. Pipette 35 microliters of this mixture over the sealing film for each cover slip, and immediately use tweezers to place the cover slips over the drop with parasites facing down.
Incubate the humid chamber on ice for five minutes, then at 37 degrees Celsius for one hour. Begin by pipetting two milliliters of denaturation solution into each well of a six well plate. Transfer the gellified cover slips containing Trypanosoma cruzi parasites into the denaturation solution and incubate with gentle agitation.
To encourage gel detachment. Using a metal spatula, carefully transfer the gel into a 1.5 milliliter microcentrifuge tube containing one milliliter of the denaturation solution. Incubate the tube with a secured cap on a heating block.
Use a P1000 pipette to aspirate the denaturation solution from the microcentrifuge tubes. With a metal spatula, transfer the gel to a Petri dish containing 10 milliliters of ultrapure water and leave it for 30 minutes. Using a three milliliter disposable Pasteur pipette, replace the ultrapure water in the dish, leaving the gel submerged overnight at room temperature.
The next day, measure the gel diameter with a caliper to calculate the expansion. The samples were visible as a planar and translucent gel that expanded 4 to 4.5 times in water. Begin the fluorescent labeling by washing the expanded gel circles containing the parasites with PBS.
Use a razor blade to cut the circles at the center into 10 by 10 millimeter squares. Now, transfer each square to a 12 well plate and incubate it in 500 microliters of the primary antibody diluted in 2%PBS and BSA, with shaking. Next, incubate the gel squares in a six well plate containing two milliliters of PBS with 0.1%polysorbate 20 for 10 minutes, with shaking.
After transferring the gel to a 12 well plate, add 500 microliters of secondary antibody in PBS with DAPI and 10 micrograms per milliliter of NHS ester conjugated to the desired fluorophore. Incubate the gel for 2.5 hours at 37 degrees Celsius under gentle agitation. After washing the gel thrice in PBS polysorbate 20 as earlier, transfer the gel to a Petri dish with ultrapure water and incubate for 30 minutes.
Place a 10 by 10 millimeter square of the expanded gel piece on a 35 millimeter bottom dish. Check the orientation of the parasites at 10 or 20X magnification. Image the samples in a confocal microscope with a 63X oil immersion objective, having 1.4 numerical aperture.
Acquire Z-stacks, the width of each Z-step, and empirically determined exposure time per pixel, then acquire the signal intensity and optimization of acquisition times using the scan zoom for effective magnification, if desired. Open the Z-stack using image processing software and group the stacked images using the group Z project option for each channel. Then, select the maximum intensity projection.
Merge images by using the Merge Channels tool. Select the color of each channel and add the scale bar using the Scale Bar tool in the processing software. The primary expansion provided an effective resolution of 70 nanometers, while the secondary expansion showed expansion factors of around 4.5.
Staining all three in vitro lifecycle stages of T.cruzi with cytoskeletal markers showed the microtubular corset and flagellar axoneme localization of the protein. Chromatin condensation was clearly distinguished when stained with DAPI. Immunolocalization of alpha tubulin markers overlapped with pan proteome labeling in epimastigotes showed that the basal body stained with anti-alpha-tubulin and FITC had divided.
Pan proteome labeling helped to identify different parasite structures at all lifecycle stages.
