Ultrastructural Expansion Microscopy in Three In Vitro Life Cycle Stages of Trypanosoma cruzi

Abstract

We describe here the application of ultrastructure expansion microscopy (U-ExM) in Trypanosoma cruzi, a technique that allows increasing the spatial resolution of a cell or tissue for microscopic imaging. This is performed by physically expanding a sample with off-the-shelf chemicals and common lab equipment.

Chagas disease is a widespread and pressing public health concern caused by T. cruzi. The disease is prevalent in Latin America and has become a significant problem in non-endemic regions due to increased migration. The transmission of T. cruzi occurs through hematophagous insect vectors belonging to the Reduviidae and Hemiptera families. Following infection, T. cruzi amastigotes multiply within the mammalian host and differentiate into trypomastigotes, the non-replicative bloodstream form. In the insect vector, trypomastigotes transform into epimastigotes and proliferate through binary fission.The differentiation between the life cycle stages requires an extensive rearrangement of the cytoskeleton and can be recreated in the lab completely using different cell culture techniques.

We describe here a detailed protocol for the application of U-ExM in three in vitro life cycle stages of Trypanosoma cruzi, focusing on optimization of the immunolocalization of cytoskeletal proteins. We also optimized the use of N-Hydroxysuccinimide ester (NHS), a pan-proteome label that has enabled us to mark different parasite structures.

Introduction

Expansion microscopy (ExM) was described for the first time in 2015 by Boyden et al.¹. It is an imaging protocol with which a conventional microscope can achieve a spatial resolution below the diffraction limit. This higher resolution is obtained because of a physical enlargement of the sample. To accomplish this, fluorescently labeled molecules are crosslinked to a hydrogel, which is subsequently expanded isotropically with water. As a result of this expansion, the signals are separated nearly isotropically in all three dimensions. This method employs low-cost chemicals and enables a spatial resolution of approximately 65 nm using conventional (confocal) microscopes, which is roughly four times better than the standard resolution of a confocal microscope (approximately 250 nm)¹.

The next milestone, that has enabled the use of expansion microscopy in many biological fields, was the adaptation of immunofluorescence labeling with conventional antibodies². Another adaptation from the initially published ExM protocol is the magnified analysis of the proteome (MAP)³. This method introduced the use of high concentrations of acrylamide and paraformaldehyde prior to sample-hydrogel immersion to prevent intra- and inter-protein crosslinking, which led to better preservation of the samples' protein content and subcellular architecture. This alternative protocol was optimized to obtain enhanced conservation of the overall ultrastructure of isolated organelles by utilizing lower concentrations of the fixative agents (formaldehyde/paraformaldehyde and acrylamide); this approach was termed ultrastructure expansion microscopy (U-ExM)⁴.

To gain even more resolution, the combination of ExM with super-resolution microscopy techniques, including stimulated emission depletion microscopy or single-molecule localization microscopy, has also been reported in order to reach resolutions below 20 nm⁵.

The use of ExM has been widely reported in the fields of neuroscience and cytoskeleton research⁶, but only a few studies have been conducted on parasitic protists. Our laboratory was the first to report the application of U-ExM in T. cruzi⁷. The foundation protocol is mainly based on the previous U-ExM reports in Toxoplasma gondii, Plasmodium ssp., and Trypanosoma brucei^8,9,10,11.

One of the greatest advantages of ExM is its modular nature, which allows great flexibility to adapt to different biological samples. The protocol can be divided into steps (such as fixation, crosslinking prevention, or gelation) that can be easily adjusted by the user to meet their experimental requirements. Additionally, this pipeline can be modified to enhance compatibility with the model organism or to achieve a specific resolution. As a result, ExM offers tremendous potential for both advanced and non-advanced optical systems, ensuring wider applications in the future.

Chagas disease, also called American trypanosomiasis, is an endemic disease in Latin America caused by Trypanosoma cruzi, a protozoan parasite. The parasite's life cycle is complex and involves two developmental stages in mammals and two in the insect host (members of the Triatominidae family), which is the biological vector of this disease. Chagas disease belongs to the group of neglected tropical diseases listed by the World Health Organization and represents a significant economic and social problem in Latin America. Epidemiological studies estimate 8 million people around the world live with Chagas disease and over 10,000 deaths per year. These numbers exemplify the significance of Chagas disease as a public health issue worldwide. The geographic distribution of Chagas disease has changed in recent decades, with many infected individuals now residing in large urban areas globally due to increased migrations, as opposed to the primarily rural areas of Latin America where it was originally found¹².

The developmental stages of T. cruzi differ throughout its life cycle, which can be replicated completely in vitro. Epimastigotes are replicative forms in the insect vector, and they have a spherical nucleus in the central region of the cell body and a bar-shaped kinetoplast (a mitochondrial DNA-containing structure unique to kinetoplastids) in the anterior region relative to the nucleus, with a free flagellum. Trypomastigotes are the infective, non-replicative form, and have an elongated nucleus, a rounded posterior kinetoplast, and a flagellum attached to the plasma membrane along the entire length of the parasite. Amastigotes are the intracellular replicative form; they have a nucleus in the central region, a rod-shaped kinetoplast in the anterior part of the cell body, and a reduced flagellum. The parasite's adaptability to different environments is a reflection of these morphological variations. It is also worth mentioning that this life cycle involves symmetrical division and different transitional developmental stages¹³. During differentiation, the trypanosomatids' cytoskeleton plays a critical role. This structure is formed by a corset of subpellicular microtubules arranged in an ordered array of stable microtubules below the plasma membrane. Also, a paraflagellar rod is present in these organisms, which is a lattice-like structure that runs parallel and is attached to the flagellar axoneme¹⁴. The precise cytoskeletal organization and nuclear structural changes along the cell cycle stages involve unique gene regulation mechanisms specific to trypanosomatids, making them interesting models for cell biology studies.

Given the small size of T. cruzi and other protozoan parasites, U-ExM presents an excellent tool for analyzing the structural features of these important pathogens. As mentioned earlier, the applicability of this technique on T. cruzi was validated for the first time by Dr. Alonso⁷. This report details a complete U-ExM protocol, with emphasis on the immunolocalization of cytoskeletal proteins during the different life cycle stages of T. cruzi. Also, we have optimized the use of N-Hydroxysuccinimide ester (NHS), a pan-proteome label that enables us to mark various parasite structures. In addition, an in vitro methodology to obtain the three stages of the parasite is described.

Protocol

NOTE: Figure 1 illustrates the complete experimental design.

Figure 1: U-ExM workflow for three in vitro life cycle stages of T. cruzi. Please click here to view a larger version of this figure.

1. Preparation of the poly-D-lysine-coated coverslips

1. Place a 10 cm x 10 cm square of sealing film in a Petri dish. Wash the coverslips by bathing them in absolute ethanol in a 35 mm glass Petri dish.
2. Remove the coverslips with tweezers from the ethanol bath and drain the excess liquid with tissue paper. Place the coverslips over the sealing film.
3. Absorb the rest of the ethanol with microscopy-grade paper. Add a 0.1% v/v solution of poly-D-lysine on the center of the coverslip and spread it with the tip to cover approximately 80% of its surface. Close the Petri dish and incubate for 1 h at 37 °C.
   NOTE: For 22 mm² coverslips, use 200 µL of poly-D-lysine solution; for 12 mm round coverslips, use 100 µL of poly-D-lysine solution.
4. Wash the coverslips with ultra-pure water three times. Use aspiration with a vacuum to remove the water between each wash. Keep at 4 °C for up to 1 week.

2. Solution preparation

1. Prepare stock solutions of 38% (w/w) sodium acrylate (SA).
   1. Slowly add 19 g of SA to 31 mL of nuclease-free water while stirring.
      NOTE: This solution is very viscous; pay attention when pipetting.
   2. Once the SA is completely dissolved, store it in a sterile container. Keep it at 4 °C and change it every 6 months.
      NOTE: SA sometimes shows signs of contamination with sodium polyacrylate depending on the brand, which is noticeable when preparing the stock solution as it turns yellowish and cloudy. Use it cautiously if this is the case.
2. Prepare a stock solution of 40% (w/v) acrylamide (AA) and a stock solution of 2% (w/v) N, N'-methylene bisacrylamide (BIS). Dissolve each compound in ultrapure water and filter with a 0.22 µm sterile syringe filter. Store in a sterile container at 4 °C.
   CAUTION: AA and BIS are highly toxic substances. Work under a fume hood and use proper protection elements (gloves, protective clothing, mask, and safety glasses).
3. Prepare the protein crosslinking prevention (CP) solution by mixing 38 µL of a 37% formaldehyde (FA) solution with 50 µL of the 40% acrylamide stock solution (step 2.2) in 912 µL of phosphate-buffered saline (PBS; Table 1) to obtain a final concentration of 1.4% formaldehyde and 2% acrylamide.
   NOTE: Always prepare the CP solution freshly and be extremely precise when pipetting. For example, use a P1000 pipette to take 900 µL of PBS and a P20 pipette to take the remaining 12 µL of PBS.
4. Prepare the monomeric solution
   NOTE: Do not use the solution immediately after being prepared; store at -20 °C at least 24 h before being used.
   1. Mix 500 µL of the 38% SA solution (step 2.1), 250 µL of the 40% AA solution, and 50 µL of the BIS solution (step 2.2), as well as 100 µL of 10x PBS (Table 1).
   2. Aliquot this final volume of 900 µL in 10x 1.5 mL microcentrifuge tubes with 90 µL of monomeric solution each. Store at -20 °C for up to 2 weeks.
      NOTE: The SA solution may stay on the tip when pipetting because it is very viscous; be careful and dispense everything.
5. Prepare the denaturing solution
   1. Mix 114.3 mL of a 350 mM sodium dodecyl sulfate (SDS) solution prepared in ultrapure water with 10 mL of a 4 M sodium chloride solution prepared in ultrapure water. Add 12 g of Tris while stirring in a 250 mL beaker.
      CAUTION: SDS is highly toxic; use it under a fume hood wearing gloves, protective clothing, a mask, and safety glasses.
   2. Adjust the pH to 9 with a concentrated solution of hydrochloric acid. Make up to 200 mL with ultrapure water and store in a sterile flask at 4 °C.
6. Prepare a 10% ammonium persulfate (APS) solution and a 10% TEMED solution.
   1. Dissolve 0.1 g of APS in 1 mL of ultrapure water.
   2. Prepare 1 mL of a 10% TEMED solution in ultrapure water.
   3. Prepare 100 µL aliquots of both solutions in 1.5 mL microcentrifuge sterile tubes and store them at -20 °C for up to 1 month.
7. Prepare the paraformaldehyde solution.
   1. Dissolve 2 g of paraformaldehyde in 40 mL of PBS while stirring at 60 °C. Add 1 M NaOH dropwise until the solution turns from white to colorless.
      CAUTION: Formaldehyde is highly toxic; use it under a fume hood wearing gloves, protective clothing, a mask, and safety glasses.
   2. Cool the solution at room temperature and adjust the pH with NaOH to 7.2 in a final volume of 50 mL. Filter with a 0.22 µm sterile syringe filter and store in a sterile container.
   3. Prepare aliquots of 1 mL in 1.5 mL sterile microcentrifuge tubes and store at -20 °C.
8. Prepare the paraformaldehyde/glutaraldehyde solution.
   1. Add 0.2 g of paraformaldehyde in 3.5 mL of ultrapure water and 50 µL of 16 M NaOH solution. Heat the solution to 60 °C to dissolve the paraformaldehyde.
   2. Cool down and add 300 µL of 70% glutaraldehyde. Bring up the volume to 5 mL with ultrapure water and finally to 10 mL with PBS.
      CAUTION: Paraformaldehyde and glutaraldehyde are highly toxic; use them under a fume hood wearing gloves, protective clothing, a mask, and safety glasses.
   3. Prepare aliquots of 1 mL in 1.5 mL sterile microcentrifuge tubes and store at -20 °C.

3. Preparation of the parasite cultures

1. Grow T. cruzi epimastigotes.
   1. Use an axenic culture in a T-25 flask (25 cm² growth area) and maintain the cultures in the logarithmic phase by sub-culturing every 48-72 h in liver infusion tryptose (LIT) medium with 10% fetal calf serum (FCS; Table 1).
   2. Ensure the cap is securely closed and keep the culture flask vertically at 28 °C for incubation. Monitor the parasite growth by cell counting in a Neubauer chamber during each subculture.
      NOTE: For the Dm28c strain used in this study, the concentration of epimastigotes in log-phase cultures is between 1-5 x 10⁷ parasites/mL.
   3. Prepare a suspension of 2 x 10⁶ epimastigotes/mL from a log-phase culture in LIT medium supplemented with 10% FCS. Centrifuge the suspension at 5,000 x g for 10 min at room temperature (RT). Wash with PBS once or twice and resuspend in 200 µL of PBS.
   4. Adhere to the round 12 mm coverslip previously coated with poly-D-lysine (section 1). Incubate at RT for 15-20 min. Continue to the crosslinking prevention step (section 4).
      NOTE: Alternatively, fix the epimastigotes with cold methanol for 7 min or paraformaldehyde/glutaraldehyde solution (step 2.8) for 10 min at RT prior to adherence of the parasites to the coverslip. It is possible to store the fixed parasites at 4 °C for up to 1 week.
2. Obtain the amastigotes from infected Vero cells.
   1. Lay a sterile 12 mm round coverslip on the bottom of a 24-well tissue culture plate. Prepare a suspension of 2 x 10⁵ Vero cells/mL in Dulbecco's modified Eagle medium (DMEM) supplemented with 2% fetal calf serum (FCS). Seed 500 µL of the suspension per well.
      NOTE: This study uses DMEM with 2% FCS to allow the cells to grow slower.
   2. Incubate overnight (ON; 12-16 h) at 37 °C and 5% CO₂ to ensure cell attachment.
   3. Following incubation, wash the cells twice using 500 µL of sterile PBS. Add T. cruzi trypomastigotes into the cells at a multiplicity of infection (MOI) of 10 in 100 µL of DMEM with 2% FCS per well, corresponding to one million trypomastigotes per well. Incubate at 37 °C and 5% CO₂ for 6 h.
   4. After incubation, rinse the plates twice with PBS. Add 500 µL of DMEM supplemented with 2% FCS. At this point, continue to the crosslinking prevention step (section 4).
      NOTE: Intracytoplasmic amastigotes will be visible through an inverted optical microscope 2 days post-infection (Supplementary Figure 1). Alternatively, fix the trypomastigotes with cold methanol for 7 min at -20 °C, or paraformaldehyde/glutaraldehyde for 10 min at RT, prior to adherence of the parasites to the coverslip.
3. Obtain trypomastigotes from infected Vero cells.
   1. Collect the supernatant of a Vero cell monolayer (30%-40% confluence) infected with trypomastigotes (MOI 1:10; ON incubation) 4 days post-infection.
      NOTE: For a T-25 flask, the initial concentration of Vero cells used is 800,000 cells, and for a T-75 flask is two million cells.
   2. Determine the trypomastigote concentration using a Neubauer chamber for cell counting. Centrifuge 4 x 10⁶ trypomastigotes at 7,000 x g at RT for 10 min.
   3. Rinse with PBS twice and resuspend in 200 µL of PBS. Apply to a round, 12 mm coverslip coated with poly-D-lysine (section 1). Incubate for 10-15 min at RT. Proceed with the crosslinking prevention step (section 4).
      ​NOTE: Alternatively, fix the trypomastigotes with cold methanol for 7 min at -20 °C or paraformaldehyde/glutaraldehyde for 10 min at RT prior to adherence of the parasites to the coverslip.

4. Performing crosslinking prevention (DAY 1)

1. Submerge the 12 mm coverslip with the adhered parasites or infected cells (facing upward) in a 24-well plate with 0.5 mL of CP solution (step 2.3) in each well.
2. Fill the empty wells with water to reduce evaporation. Seal the plate with a sealing film. Incubate for 5 h at 37 °C. This step can be extended up to an ON incubation at 4 °C.
   ​NOTE: Always submerge the coverslip in the solution; do not pipette the fixative solution over the coverslips.

5. Performing gelation of the sample

1. Assemble a humid chamber in a Petri dish with a sealing film on top of tissue paper (Figure 2A). Add water to the tissue paper and incubate at -20 °C for 20 min to cool down. Thaw a TEMED and an APS aliquot on ice for 20 min (step 2.6).
   NOTE: Do not freeze-thaw the APS more than three times.
2. Lay the cool, humid chamber on ice (Figure 2A). Take the 24-well plate prepared in section 4 out of the incubator. Aspirate the CP solution with a 3 mL Pasteur pipette, leaving some solution, otherwise the coverslips will be hard to remove.
3. Remove the 12 mm coverslips from the fixative solution with tweezers and lay them on tissue paper with the parasites facing up.
   NOTE: It is helpful to use a sterile needle to lift the coverslip and then hold it with the tweezers.
4. To an aliquot of 90 µL of monomeric solution (step 2.4), add 5 µL of TEMED and 5 µL of APS previously thawed. Mix with a vortex mixer for no more than 2-3 s; it is not necessary to close the tube with the lid.
   NOTE: Always add TEMED first and APS last. The addition of APS first and TEMED last to the monomeric solution makes the gelation process faster, giving no time to manipulate it.
5. Quickly make one drop of 35 µL over the sealing film of the humid chamber for each coverslip. Immediately pick up the coverslip with tweezers and lay over the drop (Figure 2B) with the parasites facing down.
   ​NOTE: Make a maximum of two coverslips at a time; it is crucial not to delay this step because the solution polymerizes very quickly.
6. Incubate the humid chamber for 5 min on ice and then for 1 h at 37 °C. Turn on a heating block at 95 °C to ensure the correct temperature for the next step.

Figure 2: Gelation step details. (A) Assembly of the humid chamber. (B) Dropping the coverslips onto the monomer solution with TEMED and APS for gelation. (C) Schematic representation of the gel assembled for imaging. Please click here to view a larger version of this figure.

6. Denaturing the gelified samples and performing the isotropic expansion

1. Remove the denaturation solution (step 2.5) from 4 °C. If it is precipitated, put it in a hot water bath until it dissolves completely.
2. Add 2 mL of the denaturing solution to each well of a 6-well plate. Transfer the coverslips from step 5.3 to the plate with the denaturation solution. Incubate for 15 min at RT with gentle shaking so the gel detaches from the coverslip.
3. Carefully transfer the gel (removing it from the 12 mm coverslip) with a metal spatula into a 1.5 mL sterile microcentrifuge tube with 1 mL of denaturing solution. Use cap locks to secure the tubes. Incubate for 1 h and 30 min at 95 °C in a heating block.
   NOTE: Gels start to expand during this step; be gentle when transferring the gel to the 1.5 mL microcentrifuge tube.
   CAUTION: After the incubation, the tube containing the gel is at 95 °C, which can be dangerous. Use protective gloves and let the tubes cool down before handling them to avoid burns and projections.
4. Perform the first round of expansion
   1. Aspirate the denaturing solution from the 1.5 mL microcentrifuge tube with the gel with a P1000 pipette. Transfer the gel from the 1.5 mL microcentrifuge tube to a Petri dish with 10 mL of ultrapure water for 30 min using a small spatula.
   2. Change the ultrapure water using a disposable 3 mL Pasteur pipette. Incubate ON at RT.
      NOTE: Be gentle with the gels because, after the first 30 min of incubation, they became fragile.
   3. Change the ultrapure water using a disposable 3 mL Pasteur pipette one more time.
      NOTE: Three water incubations of 30 min each are sufficient, but for practicality, it is better to leave the second incubation ON.

7. Performing fluorescence labeling of the target proteins (DAY 2)

1. Remove the water from the Petri dish with the gel with a disposable 3 mL Pasteur pipette. Measure the diameter of the gel with a caliper to calculate the expansion (between four and five times).
2. Wash twice with 10 mL of PBS for 15 min. Cut the gel with a razor blade into squares of approximately 10 mm x 10 mm in the center of the circular gel. Use one square per condition to be tested.
   NOTE: The gel shrinks after the PBS incubations. Gels can be stored in PBS at 4 °C for up to 1 week.
3. Transfer each square to a 12-well plate and incubate with 500 µL of primary antibody diluted in 2% PBS-bovine serum albumin (BSA) for 2 h and 30 min at 37 °C with shaking.
   NOTE: Alternative antibody incubation can be performed ON at 4 °C. The minimum antibody volume that can be used is 300 µL in a 24-well plate. As a general rule, use twice the concentration of antibodies used for conventional immunolabeling.
4. Wash three times with 2 mL of PBS with 0.1% polysorbate 20 for 10 min while shaking in a 6-well plate.
5. Transfer the gel to a 12-well plate and incubate with 500 µL of secondary antibody in PBS with DAPI and 10 µg/mL NHS-ester conjugated to the desired fluorophore for 2 h and 30 min at 37 °C with gentle shaking.
   NOTE: As a general rule, use twice the concentration of antibodies used for conventional immunolabeling. Alternatively, this incubation can be performed ON at 4 °C.
6. Wash three times with 2 mL of PBS with 0.1% polysorbate 20 for 10 min while shaking in a 6-well plate.
7. Transfer the gel to a Petri dish with ultrapure water. Incubate for 30 min. Change the water twice with a disposable 3 mL Pasteur pipette, as done in step 6.4.

8. Imaging and image processing (DAY 3)

1. Remove the water from the Petri dishes with a disposable 3 mL Pasteur pipette and measure the gel diameter with a caliper to calculate the expansion factor.
2. Cut a small piece of ~10 mm x 10 mm with a razor blade and place it on a 35 mm glass bottom dish.
   NOTE: As an alternative, place the gel between a slide and a coverslip (without poly-D-lysine). The slide should have two smaller slide pieces cut with a diamond knife attached on the sides to form a chamber about the thickness of the gel.
3. Check the orientation at 10x or 20x magnification. In order to focus properly, ensure that the parasites are facing the coverslip; otherwise, one has to turn the gel around and check again (Figure 2C).
4. Once the proper orientation is found, dry the remaining gel with microscopy paper. Cover the gel with a poly-D-lysine-coated coverslip.
5. Add a small drop of ultrapure water to the gel to visualize with a confocal microscope.
   NOTE: To ensure proper imaging, it is crucial to maintain the gel orientation throughout the process so that the side of the gel containing cells near its surface is facing the glass-bottom dish at the end. When using 35 mm dishes, adding poly-D-lysine to the coverslip placed in the bottom reduces the gel shifting during the image acquisition.
6. Image acquisition parameters
   1. Image the samples in a confocal microscope (Table of Materials) using a 63x oil immersion 1.4 numerical aperture (NA) objective.
   2. Acquire Z stacks, the width of each z step, and an exposure time per pixel, to be determined empirically depending on the sample, the signal intensity, and optimization of acquisition times. Use the scan zoom for effective magnification if desired.
   3. Open the Z stack using image processing software (Table of materials). For each channel, group the stacked images using the Group Z-project option. Select the maximum intensity projection.
   4. To merge images, use the Merge Channels tool and select the color of each channel. Add a scale bar using the Scale Bar tool in the processing software.
      NOTE: Alternatively, one can color each channel as desired (see examples in the representative results).

Representative Results

If the protocol has been properly executed (Figure 1), samples will be visible as a planar and translucent gel that can be expanded up to a factor of 4-4.5x in water (Figure 3A). This expansion provided an effective resolution of about 70 nm, which may vary depending on the final expansion factor and imaging system employed. After the second expansion process and image acquisition in a confocal microscope, we were able to observe expansion factors of around 4.5. To quantify this expansion, we measured the gels before and after step 7.1. Also, we labeled epimastigotes with α-tubulin antibodies in non-expanded and expanded parasites (Figure 3B).

When staining with cytoskeletal markers like anti-tubulin antibodies, we observed-in epimastigotes, trypomastigotes, and amastigotes-the correct localization of this protein in the subpellicular corset of microtubules and the flagellar axoneme, highlighting this technique's value in three in vitro life cycle stages of T. cruzi (Figure 3C). Also, the condensation state of the chromatin in the nucleus can be clearly distinguished when staining with DAPI (inset in Figure 3C).

To exemplify this optimized protocol for cytoskeletal proteins, we immunolocalized α-tubulin and the paraflagellar rod (PFR) in epimastigotes and overlapped it with pan-proteome labeling (Figure 3D). In the third panel of Figure 3D, we can observe a basal body stained with anti α-tubulin/Fluorescein isothiocyanate (FITC) that has divided (marked with arrows); this is the first step in the cell division of epimastigotes.

Finally, it is worth mentioning that when performing pan-proteome labeling, one can identify different parasite structures, such as the nucleus, flagella, kinetoplast, and flagellar pocket, among other organelles in all life cycle stages (Figure 3E).

Figure 3: Expanded gel with the parasites and edited confocal images. (A) Expanded gel measured with a caliper. (B) Non-expanded and expanded epimastigotes stained with anti-α-tubulin antibodies (grayscale; Alexa 555). Scale bar: 10 µm. (C) Epimastigotes, trypomastigotes, and amastigotes stained with anti-α-tubulin antibodies (magenta; Alexa 555) and DAPI (cyan; kDNA and nuclear DNA). Scale bar: 10 µm. Inset: zoom to a nucleus stained with DAPI. (D) Expanded epimastigotes stained with anti-α-tubulin antibodies or anti-PFR (magenta; FITC) and NHS-ester conjugated to Atto 594 (grayscale). Scale bar: 10 µm. Arrows indicate a duplicated basal body in the flagellar pocket area. (E) Epimastigotes, trypomastigotes, and uninfected and infected cells with amastigotes stained with NHS-ester conjugated to Atto 594 (grayscale). Scale bar: 10 µm. Images B-E were acquired with a confocal microscope using a 63x oil immersion 1.4 (NA) objective. Lasers used: 552 nm solid-state laser (20 mW), 405 nm diode laser (50 mW), and 488 nm solid-state laser (20 mW). Please click here to view a larger version of this figure.

Supplementary Figure 1: Visualization of intracytoplasmic amastigotes. (A) Inverted optical microscope 48 h post-infection of a Vero cell monolayer with Dm28c trypomastigotes; 40x objective. (B) Direct microscope; 60x oil objective. Abbreviations: N: nucleus of the Vero cell (marked with a dotted line). Red arrow: amastigotes. Please click here to download this File.

Table 1: Recipes for LIT medium, hemin, and PBS. Please click here to download this Table.

Discussion

Ultrastructural expansion microscopy is a technique that allows obtaining high-resolution images of biological samples by physically expanding them to several times their original size. The U-ExM protocol involves several critical steps that must be carefully executed to achieve optimal results⁴. First, the sample must be fixed with a CP agent and embedded in a swellable hydrogel matrix. The formaldehyde present in the CP solution interacts with the free covalent bonds of the acrylamide to prevent the formation of unwanted bonds with the sample. In this protocol, we recommend not to fix cells prior to the CP step as it reduces the expansion factor; however, it is necessary for some organelles, such as membrane-based organelles¹⁵. It is worth mentioning that it has been reported that an alternative to circumvent the formation of artifacts that sometimes is associated with chemical fixation is the cryofixation of samples prior to expansion¹⁶.

Next, the sample is physically expanded by applying a series of buffers that cause the hydrogel to swell and stretch. After expansion, the samples are stained with fluorescent probes and imaged using a confocal microscope. It is important to note that the choice of fixative, hydrogel matrix, and buffer conditions can greatly impact the final results, and careful optimization of these parameters is essential for obtaining high-quality images⁵. Additionally, careful attention must be paid to the imaging process, as the expanded sample can be delicate and easily damaged⁴.

The U-ExM protocol described here uses immunofluorescence staining and standard confocal microscopy, both common techniques in research labs. Using this protocol, three-dimensional reconstruction and image acquisition are straightforward, allowing for the efficient imaging of hundreds of cells. Unlike electron microscopy, which can be challenging due to difficulty in reaching or recognizing epitopes in resin-embedded sections, immunofluorescence markers work well with expanded specimens². If they do not, it could be related to the denaturation of epitopes during the SDS-based denaturation step. This could be circumvented by using a different homogenization step, such as using a denaturation buffer with guanidine hydrochloride and proteinase K. Also, the incubation temperature during this step can be lowered up to 37 °C. For example, for expanding isolated centrioles, an incubation of 30 min at 95 °C is recommended¹⁷ for mitochondrial structures to be preserved for 1 h at 70 °C¹⁵. Gelation timing is also critical, as premature gel polymerization can cause distortion, limit expansion, and result in the loss of target molecules. It is important to keep in mind that decreasing the incubation temperatures during denaturation can cause a decrease in the expansion factor or, possibly, no expansion. Regarding the problem of gel drifting, the use of poly-D-lysine-coated coverslips can help reduce this, and if it still occurs, image analysis plugins can correct any residual drift.

Although U-ExM is a powerful technique, there are several limitations that must be considered. One limitation is that the process of physically expanding the sample can cause distortion or damage to delicate structures, which may result in inaccurate or incomplete data. The process of staining the expanded sample can also be challenging, as the hydrogel matrix can interfere with the penetration of fluorescent probes into the sample. Furthermore, the imaging process can be time-consuming and computationally intensive, requiring specialized hardware and software for analysis.

U-ExM represents a significant advancement in the field of high-resolution imaging of biological samples. Additionally, it can provide a better understanding of the three-dimensional organization of biological samples, which is critical for many research applications. While other imaging techniques, such as electron microscopy, can also provide high-resolution images, U-ExM has several advantages, including the ability to visualize large specimens, as well as the potential for labeling with multiple fluorescent probes¹⁸.

U-ExM has the potential for several exciting future applications. One application is the study of complex cellular structures and interactions, such as the synapse¹⁹. Also, it is possible to label phospholipids metabolically within cellular membranes in expanded samples (termed LExM) to visualize organelle membranes with precision²⁰. It is also possible to image whole organs or organisms such as embryos or flies²¹. Recent publications in preprint format report modification of the original protocol to obtain a nanoscale resolution in optical microscopes; this technique is called one-step nanoscale expansion (ONE) microscopy²². Also, it has recently been reported as a preprint pan-expansion microscopy of tissue (pan-ExM-t), which is a fully optical mouse brain imaging method that combines 24-fold linear expansion with pan-labeling of proteins and immunolabeling of protein targets²³.

To summarize, U-ExM offers a cost-effective alternative to current super-resolution imaging and electron microscopy techniques, with the added advantage of being compatible with conventional microscopes found in most biology labs and imaging core facilities. The method can facilitate the study of nanoscale structures in trypanosomatids, allowing for the inspection of a population of cells and the imaging of an entire volume of a cell of interest at a relatively high resolution. This capability is particularly useful for studying specific cell types in transient phases of the cell cycle and the process of differentiation.

Materials

Name	Company	Catalog Number	Comments
0.22 micrometers sterile syringe filters PES	Membrane solutions	SFPES030022S
1 L beaker	Schott Duran	10005227
1.5-mL SPINWIN Micro Centrifuge Tube	Tarson	T38-500010
10 mL disposable sterile serynge	NP	66-32
10 mL serological pipette sterile	Jet Biofil	GSP211010
12-mm coverslips	Merienfeld GmbH	01 115 20	Round coverslips
12-well plates	Jet Biofil	TCP011012
22-mm coverslips	Corning	2845-22	Square coverslips
24-well plates	Jet Biofil	TCP-011-024
250 mL beaker	Schott Duran	C108.1
3 mL Pasteur pipette	Deltalab	200037
35-mm glass bottom dishes	Matsunami glass ind	D11130H
4′,6-Diamidine-2′-phenylindole dihydrochloride	Sigma Aldrich	D9542	DAPI
5 ml serological pipette sterile	Jet Biofil	GSP010005
6-well plates	Sarstedt	83.3920
Acrilamide	BioRad	1610101
Ammonium persulfate	Sigma Aldrich	A3678-25G	APS
ATTO 647 NHS ester	BOC Sciences	F10-0107	For pan-proteome labelling
Biosafty Cabinet	Telstar	Bio II A/P
Bovine Sodium Albumine	Sigma Aldrich	A7906	BSA
CO2 Incubator	Sanyo	MCO-15A
Confocal Microscope	Zeiss	LSM 880
Disposable Petridish	Tarsons	460095	90 mm diameter
DMEM, High Glucose	Thermo Fisher Cientific	12100046	Powder
Electronic digital caliper	Radar	RADAR-SLIDE-CALIPER
Ethanol Absolute	Supelco	1,00,98,31,000
Fetal Calf Serum	Internegocios SA	FCS FRA 500	Sterile and heat-inactivated
Fiji image processing package	ImageJ		doi:10.1038/nmeth.2019
Formaldehyde 37%	Sigma Aldrich	F8775	FA
Glass Petridish	Marienfeld Superior	PM-3400300	60 mm diameter
Glucosa D(+)	Cicarelli	716214
Glutaraldehyde 70%	Sigma Aldrich	G7776
Goat anti-Mouse IgG Secondary Antibody Alexa Fluor 555	Invitrogen	A-21422
Goat anti-Rabbit IgG Secondary Antibody FICT	Jackson Immunoresearch	115-095-003
Graduated cylinder	Nalgene	3663-1000
Graduated glass flask	Glassco	GL-274.202.01	100 mL
Heating Block	IBR		Made in house
Hemin	Frontier Scientific	H651-9
Hydrochloric acid 36.8-38.0%	Ciccarelli	918110
Ice bucket	Corning	1167U68
Incubator	Tecno Dalvo	TOC130
Liver Infusion	Difco	226920
Magnetic stirrer and heater	Lab companion	HP-3000
Metal spatula	SALTTECH	200MM
Metal tweezers	Marienfeld Superior	PM-6633002
Methanol absolut	Cicarelli	897110
Microcentrifuge tube 1.5 mL	Tarson	500010-N
Microscopy grade paper KimWipes	Kimtech Science	B0013HT2QW
Milli-Q water sistem	Merk Millipore	IQ-7003
mouse anti- alpha tubulin clone DM1A	Sigma Aldrich	T9026
mouse anti-PFR	Purified antibodies		Donated by Dr. Ariel Silber (USP)
N,N´-methylenbisacrilamide	ICN	193997	BIS
Na2HPO4	Cicarelli	834214
Neubauer chamber	Boeco	BOE 01
p1000 pipette	Gilson	PIPETMAN P1000
p1000 pipette tips	Tarson	TAR-521020B
p20 pipette	Gilson	PIPETMAN P20
p20 pipette tips	Tarson	TAR-527108
p200 pipette	Gilson	PIPETMAN P200
p200 pipette tips	Tarson	TAR-521010Y
Paraformaldehyde	Sigma Aldrich	P6148	PFA
pH / ORP / °C meter	HANNA Instruments	HI 2211
Poly-D-Lysine 0.1%	Sigma Aldrich	P8920
Potassium Chloride	Cicarelli	867212	KCl
Razor blade	Printex	BS 2982:1992
Sealing FIlm "Parafilm M"	Bemis	PM996
Sodium Acrilate	Sigma Aldrich	408220-25G	SA
Sodium Bicarbonate	Cicarelli	929211	NaHCO3
Sodium Chloride	Cicarelli	750214	NaCl
Sodium Dodecyl Sulfate	BioRad	1610302	SDS
Sodium Hidroxide	Merk	1-06498	NaOH
Sorvall ST 16 Centrifuge	Thermo Fisher Scientific	75004380
T-25 flasks	Corning	430639
TEMED	Invitrogen	15524-010
Tissue paper	Elite
Triptose	Merck	1106760500
Tris	BioRad	1610719
Tween-20	Biopack	2003-07	Polysorbate 20
Vaccum pump	Silfab	N33-A
Vero cells	ATCC	CRL-1587
Vortex MIxer	Dragon Lab	MX-S