Skip to main content
SpringerLink
Log in

In vitro and in vivo documentation of quantum dots labeled Trypanosoma cruzi–Rhodnius prolixus interaction using confocal microscopy

  • Original Paper
  • Published:
Parasitology Research Aims and scope Submit manuscript

Abstract

Semiconductor quantum dots (QDs) are highly fluorescent nanocrystals markers that allow long photobleaching and do not destroy the parasites. In this paper, we used fluorescent core shell quantum dots to perform studies of live parasite-vector interaction processes without any observable effect on the vitality of parasites. These nanocrystals were synthesized in aqueous medium and physiological pH, which is very important for monitoring live cells activities, and conjugated with molecules such as lectins to label specific carbohydrates involved on the parasite-vector interaction. These QDs were successfully used for the study of in vitro and in vivo interaction of Trypanosoma cruzi and the triatomine Rhodnius prolixus. These QDs allowed us to acquire real time confocal images sequences of live T. cruzi–R. prolixus interactions for an extended period, causing no damage to the cells. By zooming to the region of interest, we have been able to acquire confocal images at the three to four frames per second rate. Our results show that QDs are physiological fluorescent markers capable to label living parasites and insect vector cells. QDs can be functionalized with lectins to specifically mark surface carbohydrates on perimicrovillar membrane of R. prolixus to follow, visualize, and understand interaction between vectors and its parasites in real-time.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

References

  • Alves CR, Albuquerque-Cunha JM, Mello CB, Garcia ES, Nogueira NF, Bourguingnon SC, de Souza W, Azambuja P, Gonzalez MS (2007) Trypanosoma cruzi: attachment to perimicrovillar membrane glycoproteins of Rhodnius prolixus. Exp Parasitol 116:44–52

    Article  CAS  PubMed  Google Scholar 

  • Araújo CA, Mello CB, Jansen AM (2002) Trypanosoma cruzi I and Trypanosoma cruzi II: recognition of sugar structures by Arachis hypogaea (peanut agglutinin) lectin. J Parasitol 88:582–586

    PubMed  Google Scholar 

  • Bourguignon SC, de Souza W, Souto-Padrón T (1998) Localization of lectin-binding sites on the surface of Trypanosoma cruzi grown in chemically defined conditions. Histochem Cell Biol 110:527–534

    Article  CAS  PubMed  Google Scholar 

  • Chagas C (1909) Nova tripanosomiase humana. Mem Inst Oswaldo Cruz 1:159–281

    Google Scholar 

  • Chan WC, Nie S (1998) Quantun dot bioconjugate for ultrasensitive nonisotopic detection. Science 281:2016–2018

    Article  CAS  PubMed  Google Scholar 

  • Chan WC, Maxwell DJ, Gao X, Bailey RE, Han N, Nie S (2002) Luminescent quantum dots for multiplexed biological detection and imaging. Curr Opin Biotechnol 13:40–46

    Article  CAS  PubMed  Google Scholar 

  • Chaves C, Fontes A, Farias PMA, Santos BS, Menezes FD, Ferreira R, Cesar CL, Galembeck A, Figueiredo RCBQ (2008) Application of core-shell pegylated CdS/Cd(OH)2 quantum dots as biolabels of Trypanosoma cruzi parasites. Applied Surf Sci 255:728–730

    Article  CAS  Google Scholar 

  • Chiari E, Camargo EP (1984) Culturing and cloning of Trypanosoma cruzi. In: Morel CM (ed) Genes and antigens of parasites, (A Laboratory Manual). Fundação Oswaldo Cruz, Rio de Janeiro, pp 23–26

    Google Scholar 

  • Farias PMA, Santos BS, Menezes FD, Ferreira R, Fontes A, Carvalho HF, Romão L, Moura-Neto V, Amaral JCOF, César CL, Figueiredo RCBQ, Lorenzato FRB (2006) Quantum dots as fluorescent bio-labels in cancer diagnostic. Phys Stat sol (c) 11:4001–4008

    Article  Google Scholar 

  • Farias PMA, Santos BS, Menezes FD, Brasil JRAG, Ferreira R, Motta MA, Castro-Neto AG, Vieira AAS, Fontes A, César CL (2007) Highly fluorescent semiconductor core-shell CdTe-CdS nanocrystals for monitoring living yeast cell activity. Appl Phys A 89:957–961

    Article  Google Scholar 

  • Farias PMA, Santos BS, Thomaz AA, Ferreira R, Menezes FD, Cesar CL, Fontes A (2008) Fluorescent II – VI semiconductor Quantum Dots in living cells: Nonlinear microspectroscopy is na optical twezzers system. J Phys Chem B 112:2734–2737

    Article  CAS  PubMed  Google Scholar 

  • Feder D (2009a) Motilidade e divisão do parasita: mostra motilidade e parasitos intactos marcados com pontos quânticos CdSe, que emitem cor amarela. In: Portal de Chagas 08/1. http://www.fiocruz.br/chagas/cgi/cgilua.exe/sys/start.htm?sid=135

  • Feder D (2009b) Ensaio de interação In vitro: mostra a aderência do T. cruzi ao epitélio do intestino médio de R. prolixus marcado por pontos quânticos que emitem fluorescência verde CdTe (fluorescência confocal de 3 quadros por segundo de uma célula viva de T. cruzi). In: Portal de Chagas 08/2. http://www.fiocruz.br/chagas/cgi/cgilua.exe/sys/start.htm?sid=135

  • Ferrari BC, Veal D (2003) Analysis-only detection of Giardia by combining immunomagnetic separation and two-color flow cytometry. Cytometry A 51:79–86

    Article  PubMed  Google Scholar 

  • Gao X, Chen J, Chen J, Wu B, Chen H, Jiang X (2008) Quantum dots bearing lectin-functionalized nanoparticles as a platform for in vivo brain imaging. Bioconjugate Chem 19:2189–2195

    Article  CAS  Google Scholar 

  • Garcia ES, Ratcliffe NA, Whitten MM, Gonzalez MS, Azambuja P (2007) Exploring the role of insect host factors in the dynamics of Trypanosoma cruzi–Rhodnius prolixus interactions. Review J Insect Physiol 53:11–21

    Article  CAS  Google Scholar 

  • Goldman ER, Anderson GP, Tran PT, Mattoussi H, Charles PT, Mauro JM (2002) Conjugation of luminescent quantum dots with antibodies using an engineered adaptor protein to provide new reagents for fluoroimmunoassays. Anal Chem 74:841–847

    Article  CAS  PubMed  Google Scholar 

  • Goldman ER, Mattoussi H, Anderson GP, Medintz IL, Mauro JM (2005) Fluoroimmunoassays using antibody-conjugated quantum dots methods. Mol Biol 303:19–34

    CAS  Google Scholar 

  • Gonzalez MS, Hamedi A, Albuquerque-Cunha JM, Nogueira NFS, De Souza W, Ratcliffe NA, Azambuja P, Garcia ES, Mello CB (2006) Antiserum against perimicrovillar membranes and midgut tissue reduces the development of Trypanosoma cruzi in the insect vector, Rhodnius prolixus. Exp Parasitol 114:297–304

    Article  CAS  PubMed  Google Scholar 

  • Guevara P, Dias M, Rojas A, Crisante G, Abreu-Blanco MT, Umezawa E, Vazquez M, Levin M, Añez N, Ramirez JL (2005) Expression of fluorescent genes in Trypanosoma cruzi and Trypanosoma rangeli (Kinetoplastida: Trypanosomatidae): its application to parasite-vector biology. J Med Entomol 42:48–56

    Article  CAS  PubMed  Google Scholar 

  • Isola EL, Lammel EM, González Cappa SM (1986) Trypanosoma cruzi: differentiation after interaction of epimastigotes and Triatoma infestans intestinal homogenate. Exp Parasitol 62:329–335

    Article  CAS  PubMed  Google Scholar 

  • Jaffe CL, Grimaldi G, McMahon-Pratt D (1984) The cultivation and cloning of Leishmania. In: Morel CM (ed) Genes and antigens of parasites, a laboratory manual. Fundação Oswaldo Cruz, Rio de Janeiro, pp 48–91

    Google Scholar 

  • Jaiswall JK, Mattoussi H, Mauro JM, Simon SM (2003) Long-term multiple color imaging of live cells using quantum dot bioconjugates. Nature Biotechnol 21:47–51

    Article  Google Scholar 

  • Kollien AH, Schaub GA (2000) The development of Trypanosoma cruzi in Triatominae. Parasitol Today 16:381–387

    Article  CAS  PubMed  Google Scholar 

  • Kollien AH, Schmidt J, Schaub GA (1998) Modes of association of Trypanosoma cruzi with the intestinal tract of the vector Triatoma infestans. Acta Trop 70:127–141

    Article  CAS  PubMed  Google Scholar 

  • Lee LY, Ong SL, Hu JY, Ng WJ, Feng Y, Tan X, Wong SW (2004) Use of semiconductor quantum dots for photostable immunofluorescence labeling of Cryptosporidium parvum. Appl Environ Microbiol 70:5732–5736

    Article  CAS  PubMed  Google Scholar 

  • Mello CB, Azambuja P, Garcia ES, Ratcliffe NA (1996) Differential in vitro and in vivo behavior of three strains of Trypanosoma cruzi in the gut and hemolymph of Rhodnius prolixus. Exp Parasitol 82:112–121

    Article  CAS  PubMed  Google Scholar 

  • Mello CB, Nigam Y, Garcia ES, Azambuja P, Newton RP, Ratcliffe NA (1999) Studies on a haemolymph lectin isolated from Rhodnius prolixus and its interaction with Trypanosoma rangeli. Exp Parasitol 91:289–296

    Article  CAS  PubMed  Google Scholar 

  • Monici M (2005) Cell and tissue autofluorescence research and diagnostic applications. Biotechnol Annu Rev 11:227–256

    Article  CAS  PubMed  Google Scholar 

  • Nogueira NFS, Garcia ES, Gonzalez MS, De Souza W (1997) Effects of Azadirachtin A on the fine structure of the midgut of Rhodnius prolixus (Hemiptera: Reduviidae). J Invert Pathol 69:58–63

    Article  CAS  Google Scholar 

  • Parak WJ, Pellegrino T, Plank C (2005) Labelling of cells with quantum dots. Nanotechnol 16:R9–R21

    Article  CAS  Google Scholar 

  • Pereira ME, Andrade AF, Ribeiro JM (1981) Lectins of distinct specificity in Rhodnius prolixus interact selectively with Trypanosoma cruzi. Science 211:597–600

    Article  CAS  PubMed  Google Scholar 

  • Ratcliffe NA, Nigam Y, Mello CB, Garcia ES, Azambuja P (1996) Trypanosoma cruzi and erythrocyte agglutinins: a comparative study of occurrence and properties in the gut and hemolymph of Rhodnius prolixus. Exp Parasitol 83:83–93

    Article  CAS  PubMed  Google Scholar 

  • Souto RP, Fernández O, Macedo AM, Campbell DA, Zingales B (1996) DNA markers define two major phylogenetic lineages of Trypanosoma cruzi. Mol Biochem Parasitol 83:141–152

    Article  CAS  PubMed  Google Scholar 

  • Sweeney E, Hard TH, Gray N, Womack C, Jayson G, Hughes A, Dive C, Byers R (2008) Quantitative multiplexed quantum dots immunohistochemistry. Biochem Biophys Res Comm 374:181–186

    Article  CAS  PubMed  Google Scholar 

  • Tokumasu F, Dvorak JA (2003) Development and applications of quantum dots for immunocytochemistry of human erythrocytes. J Microsc 211:256–261

    CAS  PubMed  Google Scholar 

  • Tokumasu F, Fairhurst RM, Ostera GR, Brittain NJ, Hwang J, Wellems TE, Dvorak JA (2004) Band 3 modifications in Plasmodium falciparum-infected AA and CC erythrocytes assayed by autocorrelation analysis using quantum dots. J Cell Sci 118:1091–1098

    Article  Google Scholar 

  • Weng J, Song X, Li L, Qian H, Chen K, Xu X, Cao C, Ren J (2006) Highly luminescent CdTe quantum dots prepared in aqueous phase as an alternative fluorescent probe for cell imaging. Talanta 70:397–402

    Article  CAS  PubMed  Google Scholar 

  • Wilson R, Chen C, Ratcliffe NA (1999) Innate immunity in insects: the role of multiple, endogenous serum lectins in the recognition of foreign invaders in the cockroach, Blaberus discoidalis. J Immunol 1162:1590–1596

    Google Scholar 

  • Wojcieszyn JW, Schlegel RA, Lumley-Sapanski K, Jacobson KA (1983) Studies on the mechanism of polyethylene glycol-mediated cell fusion using fluorescent membrane and cytoplasmic probes. J Cell Biol 96:151–159

    Article  CAS  PubMed  Google Scholar 

  • Wu XY, Liu HJ, Liu JQ, Haley KN, Treadway JA, Larson JP, Ge NF, Peale F, Bruchez MP (2003) Immunofluorescent labeling of cancer marker Her2 and other cellular targets with semiconductor quantum dots. Nature Biotechnol 21:41–46

    Article  CAS  Google Scholar 

  • Zhelev Z, Ohba H, Bakalova R, Jose R, Satoshi Fukuoka S, Nagase T, Ishikawaa BY (2005) Fabrication of quantum dot–lectin conjugates as novel fluorescent probes for microscopic and flow cytometric identification of leukemia cells from normal lymphocytes. Chem Commun 15:1980–1982

    Article  Google Scholar 

Download references

Acknowledgments

We thank Leverson Lamonier from Biomedical Application of Lasers Lab group for kindly providing support with laser confocal microscope and Simone Telles from CEPOF, Catarina Macedo Lopes, Msc, Luciana Arboredo and Simone Teves from FIOCRUZ for their excellent technical assistance.

This investigation was supported by grants from Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ E-26/171.111/2005), IOC-FIOCRUZ, Rio de Janeiro and Centro de Pesquisa em Óptica e Fotônica, CEPOF, FAPESP.

Author information

Authors and Affiliations

  1. Laboratório de Biologia de Insetos, GBG, Universidade Federal Fluminense (UFF), Niterói, Rio de Janeiro, Brazil

    Denise Feder

  2. Laboratório de Transmissores de Leishmanioses, Setor de Morfologia, Ultraestrutura e Bioquímica de Artrópodes e Parasitos, IOC-FIOCRUZ, Rio de Janeiro, Brazil

    Suzete A. O. Gomes, Cecília V. Stahl & Jacenir R. Santos-Mallet

  3. Laboratório de Aplicações Biomédicas de Lasers, Instituto de Física Gleb Wataghin, Unicamp, Campinas, São Paulo, Brazil

    André A. de Thomaz, Diogo B. Almeida, Wagner M. Faustino & Carlos L. Cesar

  4. Depto de Biofísica e Radiobiologia, CCB, UFPE, Recife, Brazil

    Adriana Fontes

Authors
  1. Denise Feder
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Suzete A. O. Gomes
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. André A. de Thomaz
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Diogo B. Almeida
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Wagner M. Faustino
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Adriana Fontes
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Cecília V. Stahl
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Jacenir R. Santos-Mallet
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Carlos L. Cesar
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Denise Feder.

Additional information

Denise Feder and Suzete A. O. Gomes contributed equally to this work

Rights and permissions

Reprints and permissions

About this article

Cite this article

Feder, D., Gomes, S.A.O., de Thomaz, A.A. et al. In vitro and in vivo documentation of quantum dots labeled Trypanosoma cruzi–Rhodnius prolixus interaction using confocal microscopy. Parasitol Res 106, 85–93 (2009). https://doi.org/10.1007/s00436-009-1631-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00436-009-1631-6

Keywords

Search

Navigation