since the discovery of chagas ' disease in 1909 ( chagas , 1909 ) , major leaps in trypanosoma cruzi biology have been achieved and have resulted in gradually improving advanced techniques for direct parasite observation ( florentino et al . , 2014 ) . optical and electron microscopy ( de souza , 2008 ) are the most commonly used advanced techniques . early in vitro optical microscopy images of cultured cells infected with t. cruzi were first reported by meyer et al . barasa described its life cycle stages in mammalian cells : invasion , intracellular growth and differentiation and cell egress ( meyer et al . , 1942 ) . since then , imaging of live t. cruzi in its different evolutive forms has become routine in research laboratories . detailed information has been acquired using modern techniques , such as multidimensional confocal and electron microscopy ( florentino et al . , 2014 ) . live imaging is emerging as a powerful tool to examine how parasites interact with host cells and tissues , as well as their vectors ( amino et al . bioluminescence detection of luciferasetransfected pathogens have enabled researchers to identify infection loci and disease development in real time in experimental animal models ( amino et al . , 2005 ) . plasmodium spp was the first protozoan parasite to be successfully imaged in intravital observations at the singlecell level ( amino et al . , 2005 ) . using bioluminescence , t. cruzi has also been tracked in infected mice ( umekita et al . , 2001 , hyland et al . , 2008 , henriques et al . , 2014 , lewis et al . , 2014 ) and vectors ( henriques et al . , 2012 ) . however , intravital microscopy is limited by tissue penetration depths of up to a few hundred microns and blurring due to the overlaying connective tissue . to circumvent these limitations , some strategies can be adopted such as extracting more superficial tissues in the area of interest : for example , thinning skull without removing it and image intravascular and extravascular t. brucei through the remaining bone ( myburgh et al . , however , in order to image deeper areas , ex vivo explants of tissues and organs have been used as a tool to monitor cell migration and distribution ( bajnoff et al . , 2008 , mort et al . , 2014 ) . here , we describe a simple method to detect individual t. cruzi forms millimetres inside infected tissues and organs : slicing a specimen to gain access to the interior , then imaging  ex vivo slices . the balb / c or c57bl/6 mice infected with transfected t. cruzi trypomastigotes were sacrificed at the acute phase of infection ( 8  days ) . after organ removal and sectioning , slices were placed in a coverslip bottom dish , immersed in culture medium and imaged in a confocal microscope equipped with an environmentally controlled stage ( fig . amastigote nests of live ggfp parasites were visualized in the following selected organs : spleen , heart , intestine and liver ( fig .  1 ) . because the amastigotes were mostly immotile , optical sectioning through nests revealed the disposition of parasites and surrounding hoechstlabelled nuclei ( fig .  1 , arrowheads ) . tridimensional images of some parasite nests also revealed intermediate flagellated forms among amastigotes ( fig .  2b , arrowhead ) . live intermediate forms were also recorded in the nests within the intestinal tissue ( fig . the sectioned heart , spleen , liver and intestine of the mice infected with g gfp or cl dsred trypomastigotes ( 8  dpi ) were maintained ex vivo in dishes with culture medium and observed by confocal microscopy . a. unconventional linear amastigote arrays imaged in intestine sections of mice infected with ggfp parasites . the approximate nest dimensions on the right : 59  m ( x , y )    15  m ( z ) ; left : 117  m ( x , y )    24  m ( z ) . the approximate nest dimensions : 56  m ( x , y )    10  m ( z ) . c. threedimensional reconstruction of a ggfp amastigote heart nest . the approximate nest dimensions : 38  m    72  m    15  m . although nest morphology varied in different sections and tissues ( fig .  1 ) , singlecell linear arrangements of amastigotes formed unusually shaped agglomerates within the intestinal tissue ( fig . nests containing highly motile trypomastigotes were found in the spleen and liver ( movies s2 , s3 ) . parasites were imaged at the singlecell level within nests ranging from 10 up to almost 200  m wide , for up to 8  h ( movie s4 ) . highly motile trypomastigotes were found sometimes free in the tissues or sharing nests with other trypomastigotes , amastigotes or intermediate forms ( movie s1 ) . it is possible , however , that trypomastigotes are freed as a consequence of bladepromoted nest disruption . to our knowledge , this is the first description of t. cruzi imaging from inside infected tissues at the singlecell level . this method can provide , with good resolution , new information about parasite behaviour inside host tissues and the nature of neighbouring cells and elucidate infection mechanisms . a similar technique has brought relevant data on toxoplasma gondii intestinal infection ( coombes et al . , 2013 ) . therefore , the tool described here could be applied to examine areas with different depths in organs and tissues infected with other several pathogens , for instance , t. gondii within brain cysts or leishmania chagasi inside liver macrophages . transfected g ( tc i , gfp ) ( cruz et al . , 2012 ) and cl ( tcvi , dsred ) ( brener et al . , 1963 ) parasite strains were cycled as epimastigotes in lit medium or vero cell culture monolayers as trypomastigotes , as previously described ( cruz et al . , 2012 ) . trypanosoma expression construct ( ptrexdsrm ) containing dsred fluorescent protein was prepared using standard protocols . briefly , an open reading frame coding the monomeric variant of dsred was amplified by the polymerase chain reaction using oligonucleotides dsrmbf ( 5ggatccatggacaacaccgaggac3 ) and dsrmhr ( 5aagcttctactgggagccggagtg3 ) containing bamhi and hindiii restriction sites respectively ( lower case ) . amplicons were resolved on agarose gels , purified , cloned into pgemt easy vector ( promega corporation , fitchburg , wi , usa ) and sequenced for confirmation . the insert was subcloned into ptex vector ( kelly et al . , 1992 ) , finally , the ptexdsrm plasmid was digested with xbai and xhoi restriction enzymes , and the insert was subcloned into ptrex vector ( vazquez et al . , 1999 ) parasite transfections were performed using a gene pulser xcell electroporation system ( biorad ) as described by ( ramirez et al . , 2000 ) . briefly , 4    10 epimastigotes and 10  g of plasmid dna were mixed in 0.4  cm gap electroporation cuvettes . samples were submitted to two consecutive pulses of 450  v and 500  f . parasites were transferred to 5  ml of fresh lit medium and incubated at 26  c for 48  h before selecting for transfected parasites with 200  g / ml of g418 . parasites appears 58  days postinfection in the liver ( cruz et al . , 2012 ) . cl parasites are more infective and give rise to widespread acute murine infections ( lenzi et al . , 1996 ) . all experiments involving animal work were conducted under the brazilian national committee on ethics research ethic guidelines , which are in accordance with international standards ( cioms / oms , 1985 ) . the committee on ethics of animal experiments of the universidade federal de so paulo approved the protocol described in this study ( permit number : ceua 33710910/14 ) . during experimental procedures balb / c or c57bl/6 mice were infected using an intraperitoneal injection of tissue culturederived gfptransfected g strain ( cruz et al . , 2012 ) or dsredtransfected clstrain trypomastigotes . mice with parasitemia were monitored every 1  days as previously described ( brener , 1962 ) . one day after parasitemia peaked ( around day 8 pi for the cl strain and extended for all experiments ) , animals were sacrificed , and organs were removed . slices were cut with a sandwich of gem singleedge blades and immersed in phosphatebuffered saline containing 1  m hoechst 33342 ( invitrogen , carlsbad , ca , usa ) to label nuclei . samples were then cyanoacrylate glued to coverslips or inverted onto glassbottom dishes ( mattek corporation , ashland , ma , usa ) in rpmi1640 medium supplemented with 10% fetal bovine serum ( gibco , life technologies , brazil ) . ex vivo specimens were transferred to a stage top incubator at 37c and 5% co2 with controlled humidity ( tokai hit , japan ) on a leica sp5 ts confocal microscope . samples were imaged with a 63    1.40 oilimmersion objective using the resonant scanner ( 8000  hz ) mode for the heart , liver , spleen and intestine ( fig . transfected g ( tc i , gfp ) ( cruz et al . , 2012 ) and cl ( tcvi , dsred ) ( brener et al . , 1963 ) parasite strains were cycled as epimastigotes in lit medium or vero cell culture monolayers as trypomastigotes , as previously described ( cruz et al . , 2012 ) . trypanosoma expression construct ( ptrexdsrm ) containing dsred fluorescent protein was prepared using standard protocols . briefly , an open reading frame coding the monomeric variant of dsred was amplified by the polymerase chain reaction using oligonucleotides dsrmbf ( 5ggatccatggacaacaccgaggac3 ) and dsrmhr ( 5aagcttctactgggagccggagtg3 ) containing bamhi and hindiii restriction sites respectively ( lower case ) . amplicons were resolved on agarose gels , purified , cloned into pgemt easy vector ( promega corporation , fitchburg , wi , usa ) and sequenced for confirmation . the insert was subcloned into ptex vector ( kelly et al . , 1992 ) , finally , the ptexdsrm plasmid was digested with xbai and xhoi restriction enzymes , and the insert was subcloned into ptrex vector ( vazquez et al . , 1999 ) parasite transfections were performed using a gene pulser xcell electroporation system ( biorad ) as described by ( ramirez et al . , 2000 ) . briefly , 4    10 epimastigotes and 10  g of plasmid dna were mixed in 0.4  cm gap electroporation cuvettes . samples were submitted to two consecutive pulses of 450  v and 500  f . parasites were transferred to 5  ml of fresh lit medium and incubated at 26  c for 48  h before selecting for transfected parasites with 200  g / ml of g418 . parasites appears 58  days postinfection in the liver ( cruz et al . , 2012 ) . cl parasites are more infective and give rise to widespread acute murine infections ( lenzi et al . , 1996 ) . all experiments involving animal work were conducted under the brazilian national committee on ethics research ethic guidelines , which are in accordance with international standards ( cioms / oms , 1985 ) . the committee on ethics of animal experiments of the universidade federal de so paulo approved the protocol described in this study ( permit number : ceua 33710910/14 ) . during experimental procedures balb / c or c57bl/6 mice were infected using an intraperitoneal injection of tissue culturederived gfptransfected g strain ( cruz et al . , 2012 ) or dsredtransfected clstrain trypomastigotes . mice with parasitemia were monitored every 1  days as previously described ( brener , 1962 ) . one day after parasitemia peaked ( around day 8 pi for the cl strain and extended for all experiments ) , animals were sacrificed , and organs were removed . slices were cut with a sandwich of gem singleedge blades and immersed in phosphatebuffered saline containing 1  m hoechst 33342 ( invitrogen , carlsbad , ca , usa ) to label nuclei . samples were then cyanoacrylate glued to coverslips or inverted onto glassbottom dishes ( mattek corporation , ashland , ma , usa ) in rpmi1640 medium supplemented with 10% fetal bovine serum ( gibco , life technologies , brazil ) . ex vivo specimens were transferred to a stage top incubator at 37c and 5% co2 with controlled humidity ( tokai hit , japan ) on a leica sp5 ts confocal microscope . samples were imaged with a 63    1.40 oilimmersion objective using the resonant scanner ( 8000  hz ) mode for the heart , liver , spleen and intestine ( fig . a ) organs are placed onto a cold paraffin surface and slices cut with a sandwich of gem single edge blades . posterior immersion in cold pbs containing 1  m hoechst 33342 to label nuclei is not shown . ( b ) samples are cyanoacrylate glued to coverslips and inverted onto glassbottom dishes ( mattek corporation , usa ) in culture medium . in ( b ) an alternative method is presented in which samples are glued to a starched fabric mesh . ( c ) ex vivo specimens are placed in a stage top incubator at 37  c and 5% co2 with controlled humidity on a confocal microscope . trypanosoma cruzi epimastigotelike forms in the intestine . flagellated epimastigotelike forms observed in an ex vivo intestine section of a ggfp infected mouse at 8 dpi , acquired for 27  seconds with one second intervals . the nest dimensions are 56  m ( x , y ) x 10  m ( z ) . actively moving trypomastigotes observed in an ex vivo liver section of a ggfp infected mouse at 8 dpi , acquired for 90  minutes with one minute intervals . actively moving trypomastigotes observed in an ex vivo liver section of a cldsred infected mouse at 8 dpi , acquired for 1  minute with one second intervals . threedimensional reconstruction of amastigote forms observed in an ex vivo liver section of a ggfp infected mouse at 8 dpi , acquired for 11  hours with thirty minutes intervals .
summaryalthough imaging the live trypanosoma cruzi parasite is a routine technique in most laboratories , identification of the parasite in infected tissues and organs has been hindered by their intrinsic opaque nature . 
 we describe a simple method for in vivo observation of live singlecell trypanosoma cruzi parasites inside mammalian host tissues . 
 balb / c or c57bl/6 mice infected with dsredcl or gfpg trypomastigotes had their organs removed and sectioned with surgical blades . 
 ex vivo organ sections were observed under confocal microscopy . 
 for the first time , this procedure enabled imaging of individual amastigotes , intermediate forms and motile trypomastigotes within infected tissues of mammalian hosts .