Quantitative 3D Imaging of Trypanosoma cruzi-Infected Cells, Dormant Amastigotes, and T Cells in Intact Clarified Organs

CUBIC fixed tissues were washed with PBS to remove fixatives and then incubated with CUBIC-L cocktails, a basic buffered solution of amino alcohols that extract pigments and lipids from the tissue resulting in decolorization of tissue while maintaining tissue architecture. Grid lines in the paper can be seen through the tissues indicating an appropriate clearing of the organs (Figure 2A). After delipidation, tissues were washed and immersed in CUBIC-R+ and mounting solution for RI homogenization and imaging, respectively (Figure 2B).

A wild-type mouse was infected with tdTomato-expressing trypomastigotes pre-stained with the DiR near-infrared cyanine dye. The mouse was euthanized 15 days post-infection, and the intact heart was dissected, fixed, and cleared. LSFM imaging and 3D reconstruction allowed us to visualize tdTomato-expressing proliferating T. cruzi parasites (red). Autofluorescence levels in the red channel can be used for the correct visualization of heart structure and edges (Figure 2C i). LSFM was also useful for identifying drug-resistant dormant forms^7,8. Pre-infection staining of parasites with DiR dye allows for the tracking of dormant parasites by visualizing the parasites that had not diluted the dye through replication, as previously reported for CellTrace Violet⁷.

Dormant parasites (cyan) can be identified in the heart as depicted in the 3D enlarged insets (yellow arrows) (Figure 2C ii,iii). Z-projection fluorescence was segmented automatically to generate a reconstructed 3D surface model for spatial visualization and quantification of the total number of T. cruzi-infected cells and dormant parasites throughout the entire 3D reconstruction (Figure 2A). Analysis of the 3D surface model revealed 186 T. cruzi-infected cells with a higher proportion of infected cells in the heart atrium (123) compared with ventricles (63) and 18 dormant parasites in the whole heart (Figure 2C i). In a previous report, a similar pipeline to monitor the number of T. cruzi-infected cells and dormant parasites in clarified mice tissues after drug treatment was reported³¹. It is important to note that the estimates for numbers of dormant parasites are likely an undercount, as the dye dilution technique allows detection only of parasites that have not replicated significantly since the initial infection, but does not detect those that became dormant later in the infection following multiple rounds of division.

A similar approach was used to monitor the interaction between different T. cruzi strains in interferon (IFN)-gamma deficient mice. The production of IFN-gamma by effector T cells is essential for the immune control of T. cruzi. In IFN-gamma deficient mice, parasites proliferate with minimal immune restriction, yielding very high numbers of infected cells in organs. These immunodeficient models are useful tools for studying the efficacy of new drugs without the restriction imposed by immune recognition, and thus allow the detection of parasite relapse after treatment. IFN-gamma deficient mice were coinfected with tdTomato-expressing Colombiana (red) and GFP-expressing Brazil (blue) T. cruzi strains. The mice were euthanized 17 days post-infection, and the intact hearts were dissected, fixed, and cleared. In this immunodeficient model, host cells infected with both T. cruzi strains can be observed at various stages of parasite development, including large, medium, and small infected cells and recently burst ones. Slicing through the tissues shows abundant parasite-infected cells in the heart atria at various tissue depths (Figure 2E i–iii and Movie 1).

A cross of C57BL/6J-Tg(Cd8a*-cre)B8Asin/J and B6.Cg-Gt(ROSA)26Sor ^{tm14(CAG-tdTomato)Hze}/J mice, in which all T cells express the green fluorescent protein ZsGreen1, were used to monitor T cell recruitment in T. cruzi infected tissues. Mice were euthanized 14 days after infection with tdTomato-expressing parasites, and skeletal muscle was excised, cleared, and imaged by LSFM. ZsGreen1 expressing T cells (blue) and T. cruzi-infected cells (red) were robustly detected (Figure 2D i and Movie 2). 3D zoom-ins of the 3D reconstruction identified T cells in the region of an infected cell (Figure 2D ii). Vibratome thick sections (200-500 µm) of the same tissue allow us to visualize the interface of T cells and host infected cells by confocal microscopy (Figure 2D iii).

An alternative way to monitor inflammation foci is based on cell nuclei accumulation around T. cruzi-infected cells. A reporter mouse wherein all cell nuclei express the fluorescent tdTomato protein was used for this purpose. The nuclear tdTomato expression (red) was easily detected in the tissues after the clearing process. A nuclear reporter mouse was infected with GFP-expressing T. cruzi parasites (cyan), and 35 days post-infection, was euthanized, and skeletal muscles were excised, cleared, and imaged by LSFM (Figure 2F i–iii). Zoomed optical sections of skeletal muscle reveal increased cellularity by accumulating red nuclei along GFP-expressing parasites (Figure 2F ii). Vibratome sections of the same tissue confirm the previously described accumulation of red nuclei along GFP-expressing parasites by confocal microscopy (Figure 2F iii).

CUBIC protocol was also adapted for immunostaining and DNA labeling of intact, cleared organs and tissues infected with T. cruzi (Figure 3A). Mice were euthanized 40 days after infection with tdTomato-expressing parasites, and the hearts were dissected, fixed, and cleared. The intact cleared heart was washed and stained for 5 days with a green DNA marker, then washed again and immunostained for 7 days with antibodies against α-SMA. The samples were post-fixed, washed, RI matched, and imaged with LSFM. Simultaneous detection of multiple fluorescent signals, including nuclei, vasculature, and T. cruzi-infected cells, was possible following this protocol. α-SMA immunostaining (white) presented high signal levels revealing the intricate vasculature of the heart (Figure 3B ii and Movie 3). An optical section from deeper heart regions depicts the tissue penetration of α-SMA antibodies in the established conditions (Figure 3B v). DNA staining (blue) also exhibited good tissue penetration, fluorescence levels, and stable volumetric staining. Some areas with intense DNA labeling were identified in different heart locations (Figure 3B i) and around skeletal muscle fibers (Figure 3C i and Movie 4). A zoomed image of skeletal muscle showed an accumulation of blue nuclei in areas with few or undetectable tdTomato parasites, suggesting the recruitment of immune cells at sites of current or prior T. cruzi infection (Figure 3C ii). In other cases, infected host cells had little to no evidence of nearby cellular infiltrates (white arrows) (Figure 3C i). Vibratome sections of the same tissue as in Figure 3C ii show DNA staining of cells and tdTomato-expressing parasites by confocal microscopy (Figure 3C iii).

Previous experiments have shown that dormant parasites exhibited low or negligible expression of tdTomato or GFP reporter proteins (Figure 2C ii and iii). To improve the detection of these fluorescent proteins, cleared tissues were immunostained with anti-RFP or -GFP antibodies. Intact skeletal muscle tissues from mice infected with parasites expressing tdTomato or GFP were fixed, clarified, and immunostained with antibodies against RFP (RFP Booster) (Figure 3D) or nanobodies against GFP conjugated with Alexa Fluor 647 (GFP Booster) (Figure 3E), respectively. The samples were post-fixed, washed, RI matched, and imaged with LSFM. In both cases, the boosting of the GFP and tdTomato signals by the antibodies resulted in strong fluorescence (Figure 3D ii and 3E ii). The immunostainings using boosting antibodies represent a versatile tool that will be used to detect T. cruzi dormants in clarified whole organ and tissues and detect any underrepresented signal from RFP or GFP family member's reporter proteins.

Figure 1: Needle insertion during transcardiac perfusion. (A) A schematic representation showing the steps performed before perfusing the mouse through the heart and the correct position and direction of the perfusion needle in the left ventricle. A small incision in the right atrium was performed, and draining blood was collected (1); a butterfly needle was inserted into the heart apex. Maintain the direction so that the needle does not pierce through the septum (2). The inlet hole around the needle was sealed using gel-based glue (3). The liver is filled with blood and appears red before perfusion; however, after perfusion, it loses pigmentation and becomes pale. RA, right auricle; LA, left auricle; RV, right ventricle; LV, left ventricle. Please click here to view a larger version of this figure.

Figure 2: Visualization of T. cruzi-infected cells, dormant amastigotes, and inflammation foci in cleared organs. (A) Scheme of whole-organ T. cruzi-infected cells detection using tissue clearing, LSFM imaging, and software-assisted quantification in 3D surface models. (i) Bright-field images of the heart and skeletal muscle before (left) and after (right) clearing (scale bar: 1000 µm). (ii) Light-sheet fluorescence microscope. (iii) IFN-gamma- deficient mouse was infected with 2 x 10⁵ tdTomato-expressing trypomastigotes. At 17 days post-infection, the heart was dissected, CUBIC cleared, and LSFM imaged (scale bar: 500 µm). (iv) Z-projection fluorescence was segmented automatically to generate a reconstructed 3D surface model for spatial visualization and quantification of the number of T. cruzi-infected cells. A total of 736 T. cruzi-infected cells were detected in the whole heart (scale bar: 500 µm). (v) shows magnifications of the indicated volume in (iv), where cyan objects represent T. cruzi-infected cells (scale bar: 50 µm). (B) Protocol of CUBIC whole-organ clearing. (C) Visualization of proliferating and dormant T. cruzi parasites in transparent mouse heart. (i) A wild-type mouse was infected with 2 x 10⁵ tdTomato-expressing trypomastigotes stained with a DiR near-infrared cyanine dye. The heart was dissected, cleared, and LSFM imaged. tdTomato-expressing proliferating (red) and dormant (cyan) T. cruzi parasites can be identified. Autofluorescence levels were maintained to allow for correct visualization of the heart structure. A mouse was killed 15 days post-infection based on the peak of parasite-infected cells and dormant amastigotes found in previous works (scale bar: 400 µm). (ii) and (iii) show magnifications of the indicated volume in (i), where yellow arrows indicate dormant parasites (cyan) (scale bar: 200 µm). (D) Detection of T cell recruitment in infected CD8 reporter mouse. (i) Reporter mouse was infected with 2 x 10⁵ tdTomato-expressing trypomastigotes, and skeletal muscle was dissected, cleared, and LSFM imaged 14 days post-infection, a time point at which substantial cell responses are detected. ZsGreen1 expressing T cells (blue) as well as T. cruzi-infected cells (red) were visualized (scale bar: 400 µm) (see also Movie 2). (ii) shows magnifications of the indicated volume in (i) (scale bar: 50 µm). (iii) depicts T cell accumulation surrounding a T. cruzi-infected cell in a tissue section (200 µm) of the same organ (scale bar: 8 µm). (E) Interaction between different T. cruzi strains. (i) IFN-gamma deficient mice were coinfected with 2 x 10⁵ tdTomato-expressing Colombiana (red) and GFP-expressing Brazil (blue) T. cruzi strains. The mice were euthanized, and the intact hearts were dissected, fixed, and cleared at 17 days post-infection (scale bar: 400 µm). (ii) shows magnifications of the indicated volume in (i) (scale bar: 250 µm). (iii) shows a magnified optical section revealing cells infected with Colombiana (red) and Brazil (blue) T. cruzi strains (scale bar: 150 µm). (F) Visualization of cellularity along infected cells using nuclei reporter mouse. (i) A nuclear reporter mouse was infected with 2 x 10⁵ GFP-expressing trypomastigotes, and skeletal muscle was dissected, cleared, and LSFM imaged at 35 days post-infection. Nuclear tdTomato expression of the host cells (red), as well as GFP parasite expression (cyan), were detected (scale bar: 400 µm). (ii) shows a magnified optical section revealing accumulation of red nuclei along GFP-expressing parasites (scale bar: 90 µm). (iii) confirms increased cellularity by confocal imaging of tissue sections from the same organ (scale bar: 8 µm). Please click here to view a larger version of this figure.

Figure 3: Labeling of cleared T. cruzi-infected organs with antibodies and nuclearstains. (A) CUBIC protocol for tissue clearing, 3D immunostaining, and DNA labeling of entire organs. (B) Labeling of mouse heart for vasculature and DNA detection (see also Movie 3). (i–iii) A wild-type mouse was infected with 2 x 10⁵ tdTomato-expressing parasites. The mouse was euthanized 40 days after infection, and the heart was dissected, fixed, and cleared. The cleared heart was labeled with a DNA marker and immunostained with antibodies against α-SMA. Simultaneous detection of cell nuclei (blue), vasculature (white), and T. cruzi-infected cells (red) were possible. The mouse was killed at this time post-infection when tissue and special vasculature remodeling can be assessed (scale bar: 400 µm). (iv) shows a magnified volume from deep heart regions of (iii) depicting cell nuclei, vasculature, and infected cells (scale bar: 90 µm). (v) depicts an optical section obtained of (iii) (scale bar: 90 µm). (C) Increased cellularity visualized by DNA labeling of whole cleared skeletal muscle. (i) Skeletal muscle tissue from the previous experiment was DNA stained, revealing areas with intense nuclear labeling (blue) as well as T. cruzi-infected cells (red) (scale bar: 200 µm) (see also Movie 4). (ii) reveals an intense accumulation of blue nuclei in areas with lower to no detection of tdTomato parasite (scale bar: 100 µm). (iii) high magnification (60x) confocal imaging identifies DNA labeling of cells and parasites in tissue sections (scale bar: 10 µm). (D) Boosting of tdTomato parasite reporter markers in whole cleared skeletal muscle. (i–iii) Intact skeletal muscle tissues from mice infected with 2 x 10⁵ parasites expressing tdTomato were fixed, clarified, and immunostained with antibodies against RFP (RFP Booster). An intense and uniform fluorescent signal was obtained in the far-red channel after RFP boosting (green) of the tdTomato signal (red) (scale bar: 100 µm). (E) Boosting of GFP parasite reporter markers using anti-GFP nanobodies. (i–iii) Skeletal muscle tissues from mice infected with 2 x 10⁵ parasites expressing GFP were fixed, clarified, and immunostained with Alexa Fluor 647-conjugated nanobodies against GFP (GFP Booster). A strong fluorescent signal was obtained in the far-red channel after GFP boosting (magenta) of the GFP fluorescence (cyan). Mouse from (D) and (E) were killed 30 days post-infection because parasite loads reach a peak at this time point, and parasite-infected cells can be easily detected in skeletal muscle (scale bar: 100 µm). Please click here to view a larger version of this figure.

Movie 1: T. cruzi infection of the heart and skeletal muscle in IFN-gamma deficient mice. 3D reconstructions of CUBIC-clarified tissues of IFN-gamma deficient mice coinfected with 2 x 10⁵ tdTomato-expressing Colombiana (red) and GFP-expressing Brazil (blue) T. cruzi strains at day 17 post-infection. A total of 1151 individual slices of the heart and 559 of the skeletal muscle were acquired via LSFM. Comparable imaging results were achieved in three independent animals. Please click here to download this Movie.

Movie 2: Detection of T cell recruitment in T. cruzi-infected CD8 reporter mouse. 3D visualization of a CUBIC-clarified skeletal muscle from a CD8 reporter mouse infected with 2 x 10⁵ Tdtomato-expressing Colombiana T. cruzi strain (red) and killed at 14 days post-infection. A total of 668 individual slices of the skeletal muscle were imaged by LSFM. ZsGreen1 expressing T cells (blue) as well as T. cruzi-infected cells (red) were visualized. Comparable imaging results were achieved in two animals. Please click here to download this Movie.

Movie 3: Immunodetection of vasculature in T. cruzi-infected and cleared heart. 3D reconstruction of a CUBIC-clarified heart of wild-type mice infected with 2 x 10⁵ tdTomato-expressing Colombiana T. cruzi and immunostained with antibodies against α-SMA (blue) at 40 days post-infection. Slicing through the heart reveals the intricate vasculature of the heart and the tissue penetration of the α-SMA antibodies. Parasite infected cells could be observed as bright red spots in the heart atria (red, right). Comparable imaging results were achieved in two animals. Please click here to download this Movie.

Movie 4: Whole-organDNA staining reveals areas with increased cellularity. 3D reconstruction of the CUBIC-clarified skeletal muscle of wild-type mouse at 40 days post-infection infected with 2 x 10⁵ tdTomato-expressing Colombiana T. cruzi strain. The whole quadriceps muscle area was stained with a green nuclear dye. T. cruzi-infected cells (red) and the nuclei of every cell in the tissue (blue) were visualized. Zoom-ins of the 3D reconstruction reveals accumulation of blue nuclei along areas with few or no detection of tdTomato parasites. Comparable imaging results were achieved in two animals. Please click here to download this Movie.

Supplementary File 1: Composition of the antibody staining solutions used in the study. Please click here to download this File.