This study shows a detailed protocol to perform ultrastructure expansion microscopy in three in vitro life cycle stages of Trypanosoma cruzi, the pathogen responsible for Chagas disease. We include the optimized technique for cytoskeletal proteins and pan-proteome labeling.
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.
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 brucei8,9,10,11.
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.
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
2. Solution preparation
3. Preparation of the parasite cultures
4. Performing crosslinking prevention (DAY 1)
5. Performing gelation of the sample
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
7. Performing fluorescence labeling of the target proteins (DAY 2)
8. Imaging and image processing (DAY 3)
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.
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.
The authors have nothing to disclose.
We thank Dolores Campos for assisting with Vero cell culture and Romina Manarin for assisting with T. cruzi culture. This work was supported by Agencia Nacional de Promoción Científica y Tecnológica, Ministerio de Ciencia e Innovación Productiva from Argentina (PICT2019-0526), Consejo Nacional de Investigaciones Científicas y Técnicas (PIBAA 1242), and Research Council United Kingdom [MR/P027989/1].
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 |
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