Abstract

Structural details of spermatozoa are interesting from the perspectives of fundamental biology and growing reproductive health problems. Studies of nanostructural details of these extremely motile cells have been limited to fixed cells, largely using electron microscopy. Here we provide the protocols for and demonstrate live-cell multi-color super-resolution imaging of human spermatozoa using structured illumination microscopy (SIM). By using patches of agarose for immobilization, we achieved four-channel 3D SIM imaging of the plasma membrane, nucleus, mitochondria and microtubulin in the same living sperm cells. We expect that high-resolution imaging of living spermatozoa will be implemented for research on fundamental cellular mechanisms together with morphological aberrations involved in male infertility for a future improved cell selection process in in vitro fertilization treatments.

© 2018 Optical Society of America under the terms of the OSA Open Access Publishing Agreement

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  1. G. Cavallini, “Male idiopathic oligoasthenoteratozoospermia,” Asian J. Androl. 8(2), 143–157 (2006).
    [Crossref] [PubMed]
  2. N. Kumar and A. K. Singh, “Trends of male factor infertility, an important cause of infertility: A review of literature,” J. Hum. Reprod. Sci. 8(4), 191–196 (2015).
    [Crossref] [PubMed]
  3. S. Ozkavukcu, E. Erdemli, A. Isik, D. Oztuna, and S. Karahuseyinoglu, “Effects of cryopreservation on sperm parameters and ultrastructural morphology of human spermatozoa,” J. Assist. Reprod. Genet. 25(8), 403–411 (2008).
    [Crossref] [PubMed]
  4. C. Barthelemy, D. Royere, S. Hammahah, C. Lebos, M. J. Tharanne, and J. Lansac, “Ultrastructural changes in membranes and acrosome of human sperm during cryopreservation,” Arch. Androl. 25(1), 29–40 (1990).
    [Crossref] [PubMed]
  5. N. Naha, R. B. Bhar, A. Mukherjee, and A. R. Chowdhury, “Structural alteration of spermatozoa in the persons employed in lead acid battery factory,” Indian J. Physiol. Pharmacol. 49(2), 153–162 (2005).
    [PubMed]
  6. W. Asghar, H. Shafiee, V. Velasco, V. R. Sah, S. Guo, R. El Assal, F. Inci, A. Rajagopalan, M. Jahangir, R. M. Anchan, G. L. Mutter, M. Ozkan, C. S. Ozkan, and U. Demirci, “Toxicology Study of Single-walled Carbon Nanotubes and Reduced Graphene Oxide in Human Sperm,” Sci. Rep. 6(1), 30270 (2016).
    [Crossref] [PubMed]
  7. D. Lacy, A. J. Pettitt, J. M. Pettitt, and B. S. Martin, “Application of scanning electron microscopy to semen analysis of the sub-fertile man utilising data obtained by transmission electron microscopy as an aid to interpretation,” Micron (1969) 5(2), 135–173 (1974).
    [Crossref]
  8. B. Baccetti, S. Capitani, G. Collodel, G. Di Cairano, L. Gambera, E. Moretti, and P. Piomboni, “Genetic sperm defects and consanguinity,” Hum. Reprod. 16(7), 1365–1371 (2001).
    [Crossref] [PubMed]
  9. E. H. Chemes and Y. V. Rawe, “Sperm pathology: a step beyond descriptive morphology. Origin, characterization and fertility potential of abnormal sperm phenotypes in infertile men,” Hum. Reprod. Update 9(5), 405–428 (2003).
    [Crossref] [PubMed]
  10. A. J. Hobro and N. I. Smith, “An evaluation of fixation methods: Spatial and compositional cellular changes observed by Raman imaging,” Vib. Spectrosc. 91, 31–45 (2017).
    [Crossref]
  11. J. Kiernan, Formaldehyde, Formalin, Paraformaldehyde And Glutaraldehyde: What They Are And What They Do (2000).
  12. C. G. Galbraith and J. A. Galbraith, “Super-resolution microscopy at a glance,” J. Cell Sci. 124(10), 1607–1611 (2011).
    [Crossref] [PubMed]
  13. R. Heintzmann and T. Huser, “Super-Resolution Structured Illumination Microscopy,” Chem. Rev. 117(23), 13890–13908 (2017).
    [Crossref] [PubMed]
  14. E. Moretti and G. Collodel, Electron Microscopy in the Study of Human Sperm Pathologies (2012).
  15. V. Y. Rawe, G. D. Galaverna, A. A. Acosta, S. B. Olmedo, and H. E. Chemes, “Incidence of tail structure distortions associated with dysplasia of the fibrous sheath in human spermatozoa,” Hum. Reprod. 16(5), 879–886 (2001).
    [Crossref] [PubMed]
  16. F. Pelliccione, A. Micillo, G. Cordeschi, A. D’Angeli, S. Necozione, L. Gandini, A. Lenzi, F. Francavilla, and S. Francavilla, “Altered ultrastructure of mitochondrial membranes is strongly associated with unexplained asthenozoospermia,” Fertil. Steril. 95(2), 641–646 (2011).
    [Crossref] [PubMed]
  17. W. V. Holt, F. Shenfield, T. Leonard, T. D. Hartman, R. D. North, and H. D. Moore, “The value of sperm swimming speed measurements in assessing the fertility of human frozen semen,” Hum. Reprod. 4(3), 292–297 (1989).
    [Crossref] [PubMed]
  18. J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, J.-Y. Tinevez, D. J. White, V. Hartenstein, K. Eliceiri, P. Tomancak, and A. Cardona, “Fiji: an open-source platform for biological-image analysis,” Nat. Methods 9(7), 676–682 (2012).
    [Crossref] [PubMed]
  19. G. Joyce, B. D. Robertson, and K. J. Williams, “A modified agar pad method for mycobacterial live-cell imaging,” BMC Res. Notes 4(1), 73 (2011).
    [Crossref] [PubMed]

2017 (2)

A. J. Hobro and N. I. Smith, “An evaluation of fixation methods: Spatial and compositional cellular changes observed by Raman imaging,” Vib. Spectrosc. 91, 31–45 (2017).
[Crossref]

R. Heintzmann and T. Huser, “Super-Resolution Structured Illumination Microscopy,” Chem. Rev. 117(23), 13890–13908 (2017).
[Crossref] [PubMed]

2016 (1)

W. Asghar, H. Shafiee, V. Velasco, V. R. Sah, S. Guo, R. El Assal, F. Inci, A. Rajagopalan, M. Jahangir, R. M. Anchan, G. L. Mutter, M. Ozkan, C. S. Ozkan, and U. Demirci, “Toxicology Study of Single-walled Carbon Nanotubes and Reduced Graphene Oxide in Human Sperm,” Sci. Rep. 6(1), 30270 (2016).
[Crossref] [PubMed]

2015 (1)

N. Kumar and A. K. Singh, “Trends of male factor infertility, an important cause of infertility: A review of literature,” J. Hum. Reprod. Sci. 8(4), 191–196 (2015).
[Crossref] [PubMed]

2012 (1)

J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, J.-Y. Tinevez, D. J. White, V. Hartenstein, K. Eliceiri, P. Tomancak, and A. Cardona, “Fiji: an open-source platform for biological-image analysis,” Nat. Methods 9(7), 676–682 (2012).
[Crossref] [PubMed]

2011 (3)

G. Joyce, B. D. Robertson, and K. J. Williams, “A modified agar pad method for mycobacterial live-cell imaging,” BMC Res. Notes 4(1), 73 (2011).
[Crossref] [PubMed]

F. Pelliccione, A. Micillo, G. Cordeschi, A. D’Angeli, S. Necozione, L. Gandini, A. Lenzi, F. Francavilla, and S. Francavilla, “Altered ultrastructure of mitochondrial membranes is strongly associated with unexplained asthenozoospermia,” Fertil. Steril. 95(2), 641–646 (2011).
[Crossref] [PubMed]

C. G. Galbraith and J. A. Galbraith, “Super-resolution microscopy at a glance,” J. Cell Sci. 124(10), 1607–1611 (2011).
[Crossref] [PubMed]

2008 (1)

S. Ozkavukcu, E. Erdemli, A. Isik, D. Oztuna, and S. Karahuseyinoglu, “Effects of cryopreservation on sperm parameters and ultrastructural morphology of human spermatozoa,” J. Assist. Reprod. Genet. 25(8), 403–411 (2008).
[Crossref] [PubMed]

2006 (1)

G. Cavallini, “Male idiopathic oligoasthenoteratozoospermia,” Asian J. Androl. 8(2), 143–157 (2006).
[Crossref] [PubMed]

2005 (1)

N. Naha, R. B. Bhar, A. Mukherjee, and A. R. Chowdhury, “Structural alteration of spermatozoa in the persons employed in lead acid battery factory,” Indian J. Physiol. Pharmacol. 49(2), 153–162 (2005).
[PubMed]

2003 (1)

E. H. Chemes and Y. V. Rawe, “Sperm pathology: a step beyond descriptive morphology. Origin, characterization and fertility potential of abnormal sperm phenotypes in infertile men,” Hum. Reprod. Update 9(5), 405–428 (2003).
[Crossref] [PubMed]

2001 (2)

B. Baccetti, S. Capitani, G. Collodel, G. Di Cairano, L. Gambera, E. Moretti, and P. Piomboni, “Genetic sperm defects and consanguinity,” Hum. Reprod. 16(7), 1365–1371 (2001).
[Crossref] [PubMed]

V. Y. Rawe, G. D. Galaverna, A. A. Acosta, S. B. Olmedo, and H. E. Chemes, “Incidence of tail structure distortions associated with dysplasia of the fibrous sheath in human spermatozoa,” Hum. Reprod. 16(5), 879–886 (2001).
[Crossref] [PubMed]

1990 (1)

C. Barthelemy, D. Royere, S. Hammahah, C. Lebos, M. J. Tharanne, and J. Lansac, “Ultrastructural changes in membranes and acrosome of human sperm during cryopreservation,” Arch. Androl. 25(1), 29–40 (1990).
[Crossref] [PubMed]

1989 (1)

W. V. Holt, F. Shenfield, T. Leonard, T. D. Hartman, R. D. North, and H. D. Moore, “The value of sperm swimming speed measurements in assessing the fertility of human frozen semen,” Hum. Reprod. 4(3), 292–297 (1989).
[Crossref] [PubMed]

1974 (1)

D. Lacy, A. J. Pettitt, J. M. Pettitt, and B. S. Martin, “Application of scanning electron microscopy to semen analysis of the sub-fertile man utilising data obtained by transmission electron microscopy as an aid to interpretation,” Micron (1969) 5(2), 135–173 (1974).
[Crossref]

Acosta, A. A.

V. Y. Rawe, G. D. Galaverna, A. A. Acosta, S. B. Olmedo, and H. E. Chemes, “Incidence of tail structure distortions associated with dysplasia of the fibrous sheath in human spermatozoa,” Hum. Reprod. 16(5), 879–886 (2001).
[Crossref] [PubMed]

Anchan, R. M.

W. Asghar, H. Shafiee, V. Velasco, V. R. Sah, S. Guo, R. El Assal, F. Inci, A. Rajagopalan, M. Jahangir, R. M. Anchan, G. L. Mutter, M. Ozkan, C. S. Ozkan, and U. Demirci, “Toxicology Study of Single-walled Carbon Nanotubes and Reduced Graphene Oxide in Human Sperm,” Sci. Rep. 6(1), 30270 (2016).
[Crossref] [PubMed]

Arganda-Carreras, I.

J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, J.-Y. Tinevez, D. J. White, V. Hartenstein, K. Eliceiri, P. Tomancak, and A. Cardona, “Fiji: an open-source platform for biological-image analysis,” Nat. Methods 9(7), 676–682 (2012).
[Crossref] [PubMed]

Asghar, W.

W. Asghar, H. Shafiee, V. Velasco, V. R. Sah, S. Guo, R. El Assal, F. Inci, A. Rajagopalan, M. Jahangir, R. M. Anchan, G. L. Mutter, M. Ozkan, C. S. Ozkan, and U. Demirci, “Toxicology Study of Single-walled Carbon Nanotubes and Reduced Graphene Oxide in Human Sperm,” Sci. Rep. 6(1), 30270 (2016).
[Crossref] [PubMed]

Baccetti, B.

B. Baccetti, S. Capitani, G. Collodel, G. Di Cairano, L. Gambera, E. Moretti, and P. Piomboni, “Genetic sperm defects and consanguinity,” Hum. Reprod. 16(7), 1365–1371 (2001).
[Crossref] [PubMed]

Barthelemy, C.

C. Barthelemy, D. Royere, S. Hammahah, C. Lebos, M. J. Tharanne, and J. Lansac, “Ultrastructural changes in membranes and acrosome of human sperm during cryopreservation,” Arch. Androl. 25(1), 29–40 (1990).
[Crossref] [PubMed]

Bhar, R. B.

N. Naha, R. B. Bhar, A. Mukherjee, and A. R. Chowdhury, “Structural alteration of spermatozoa in the persons employed in lead acid battery factory,” Indian J. Physiol. Pharmacol. 49(2), 153–162 (2005).
[PubMed]

Capitani, S.

B. Baccetti, S. Capitani, G. Collodel, G. Di Cairano, L. Gambera, E. Moretti, and P. Piomboni, “Genetic sperm defects and consanguinity,” Hum. Reprod. 16(7), 1365–1371 (2001).
[Crossref] [PubMed]

Cardona, A.

J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, J.-Y. Tinevez, D. J. White, V. Hartenstein, K. Eliceiri, P. Tomancak, and A. Cardona, “Fiji: an open-source platform for biological-image analysis,” Nat. Methods 9(7), 676–682 (2012).
[Crossref] [PubMed]

Cavallini, G.

G. Cavallini, “Male idiopathic oligoasthenoteratozoospermia,” Asian J. Androl. 8(2), 143–157 (2006).
[Crossref] [PubMed]

Chemes, E. H.

E. H. Chemes and Y. V. Rawe, “Sperm pathology: a step beyond descriptive morphology. Origin, characterization and fertility potential of abnormal sperm phenotypes in infertile men,” Hum. Reprod. Update 9(5), 405–428 (2003).
[Crossref] [PubMed]

Chemes, H. E.

V. Y. Rawe, G. D. Galaverna, A. A. Acosta, S. B. Olmedo, and H. E. Chemes, “Incidence of tail structure distortions associated with dysplasia of the fibrous sheath in human spermatozoa,” Hum. Reprod. 16(5), 879–886 (2001).
[Crossref] [PubMed]

Chowdhury, A. R.

N. Naha, R. B. Bhar, A. Mukherjee, and A. R. Chowdhury, “Structural alteration of spermatozoa in the persons employed in lead acid battery factory,” Indian J. Physiol. Pharmacol. 49(2), 153–162 (2005).
[PubMed]

Collodel, G.

B. Baccetti, S. Capitani, G. Collodel, G. Di Cairano, L. Gambera, E. Moretti, and P. Piomboni, “Genetic sperm defects and consanguinity,” Hum. Reprod. 16(7), 1365–1371 (2001).
[Crossref] [PubMed]

E. Moretti and G. Collodel, Electron Microscopy in the Study of Human Sperm Pathologies (2012).

Cordeschi, G.

F. Pelliccione, A. Micillo, G. Cordeschi, A. D’Angeli, S. Necozione, L. Gandini, A. Lenzi, F. Francavilla, and S. Francavilla, “Altered ultrastructure of mitochondrial membranes is strongly associated with unexplained asthenozoospermia,” Fertil. Steril. 95(2), 641–646 (2011).
[Crossref] [PubMed]

D’Angeli, A.

F. Pelliccione, A. Micillo, G. Cordeschi, A. D’Angeli, S. Necozione, L. Gandini, A. Lenzi, F. Francavilla, and S. Francavilla, “Altered ultrastructure of mitochondrial membranes is strongly associated with unexplained asthenozoospermia,” Fertil. Steril. 95(2), 641–646 (2011).
[Crossref] [PubMed]

Demirci, U.

W. Asghar, H. Shafiee, V. Velasco, V. R. Sah, S. Guo, R. El Assal, F. Inci, A. Rajagopalan, M. Jahangir, R. M. Anchan, G. L. Mutter, M. Ozkan, C. S. Ozkan, and U. Demirci, “Toxicology Study of Single-walled Carbon Nanotubes and Reduced Graphene Oxide in Human Sperm,” Sci. Rep. 6(1), 30270 (2016).
[Crossref] [PubMed]

Di Cairano, G.

B. Baccetti, S. Capitani, G. Collodel, G. Di Cairano, L. Gambera, E. Moretti, and P. Piomboni, “Genetic sperm defects and consanguinity,” Hum. Reprod. 16(7), 1365–1371 (2001).
[Crossref] [PubMed]

El Assal, R.

W. Asghar, H. Shafiee, V. Velasco, V. R. Sah, S. Guo, R. El Assal, F. Inci, A. Rajagopalan, M. Jahangir, R. M. Anchan, G. L. Mutter, M. Ozkan, C. S. Ozkan, and U. Demirci, “Toxicology Study of Single-walled Carbon Nanotubes and Reduced Graphene Oxide in Human Sperm,” Sci. Rep. 6(1), 30270 (2016).
[Crossref] [PubMed]

Eliceiri, K.

J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, J.-Y. Tinevez, D. J. White, V. Hartenstein, K. Eliceiri, P. Tomancak, and A. Cardona, “Fiji: an open-source platform for biological-image analysis,” Nat. Methods 9(7), 676–682 (2012).
[Crossref] [PubMed]

Erdemli, E.

S. Ozkavukcu, E. Erdemli, A. Isik, D. Oztuna, and S. Karahuseyinoglu, “Effects of cryopreservation on sperm parameters and ultrastructural morphology of human spermatozoa,” J. Assist. Reprod. Genet. 25(8), 403–411 (2008).
[Crossref] [PubMed]

Francavilla, F.

F. Pelliccione, A. Micillo, G. Cordeschi, A. D’Angeli, S. Necozione, L. Gandini, A. Lenzi, F. Francavilla, and S. Francavilla, “Altered ultrastructure of mitochondrial membranes is strongly associated with unexplained asthenozoospermia,” Fertil. Steril. 95(2), 641–646 (2011).
[Crossref] [PubMed]

Francavilla, S.

F. Pelliccione, A. Micillo, G. Cordeschi, A. D’Angeli, S. Necozione, L. Gandini, A. Lenzi, F. Francavilla, and S. Francavilla, “Altered ultrastructure of mitochondrial membranes is strongly associated with unexplained asthenozoospermia,” Fertil. Steril. 95(2), 641–646 (2011).
[Crossref] [PubMed]

Frise, E.

J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, J.-Y. Tinevez, D. J. White, V. Hartenstein, K. Eliceiri, P. Tomancak, and A. Cardona, “Fiji: an open-source platform for biological-image analysis,” Nat. Methods 9(7), 676–682 (2012).
[Crossref] [PubMed]

Galaverna, G. D.

V. Y. Rawe, G. D. Galaverna, A. A. Acosta, S. B. Olmedo, and H. E. Chemes, “Incidence of tail structure distortions associated with dysplasia of the fibrous sheath in human spermatozoa,” Hum. Reprod. 16(5), 879–886 (2001).
[Crossref] [PubMed]

Galbraith, C. G.

C. G. Galbraith and J. A. Galbraith, “Super-resolution microscopy at a glance,” J. Cell Sci. 124(10), 1607–1611 (2011).
[Crossref] [PubMed]

Galbraith, J. A.

C. G. Galbraith and J. A. Galbraith, “Super-resolution microscopy at a glance,” J. Cell Sci. 124(10), 1607–1611 (2011).
[Crossref] [PubMed]

Gambera, L.

B. Baccetti, S. Capitani, G. Collodel, G. Di Cairano, L. Gambera, E. Moretti, and P. Piomboni, “Genetic sperm defects and consanguinity,” Hum. Reprod. 16(7), 1365–1371 (2001).
[Crossref] [PubMed]

Gandini, L.

F. Pelliccione, A. Micillo, G. Cordeschi, A. D’Angeli, S. Necozione, L. Gandini, A. Lenzi, F. Francavilla, and S. Francavilla, “Altered ultrastructure of mitochondrial membranes is strongly associated with unexplained asthenozoospermia,” Fertil. Steril. 95(2), 641–646 (2011).
[Crossref] [PubMed]

Guo, S.

W. Asghar, H. Shafiee, V. Velasco, V. R. Sah, S. Guo, R. El Assal, F. Inci, A. Rajagopalan, M. Jahangir, R. M. Anchan, G. L. Mutter, M. Ozkan, C. S. Ozkan, and U. Demirci, “Toxicology Study of Single-walled Carbon Nanotubes and Reduced Graphene Oxide in Human Sperm,” Sci. Rep. 6(1), 30270 (2016).
[Crossref] [PubMed]

Hammahah, S.

C. Barthelemy, D. Royere, S. Hammahah, C. Lebos, M. J. Tharanne, and J. Lansac, “Ultrastructural changes in membranes and acrosome of human sperm during cryopreservation,” Arch. Androl. 25(1), 29–40 (1990).
[Crossref] [PubMed]

Hartenstein, V.

J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, J.-Y. Tinevez, D. J. White, V. Hartenstein, K. Eliceiri, P. Tomancak, and A. Cardona, “Fiji: an open-source platform for biological-image analysis,” Nat. Methods 9(7), 676–682 (2012).
[Crossref] [PubMed]

Hartman, T. D.

W. V. Holt, F. Shenfield, T. Leonard, T. D. Hartman, R. D. North, and H. D. Moore, “The value of sperm swimming speed measurements in assessing the fertility of human frozen semen,” Hum. Reprod. 4(3), 292–297 (1989).
[Crossref] [PubMed]

Heintzmann, R.

R. Heintzmann and T. Huser, “Super-Resolution Structured Illumination Microscopy,” Chem. Rev. 117(23), 13890–13908 (2017).
[Crossref] [PubMed]

Hobro, A. J.

A. J. Hobro and N. I. Smith, “An evaluation of fixation methods: Spatial and compositional cellular changes observed by Raman imaging,” Vib. Spectrosc. 91, 31–45 (2017).
[Crossref]

Holt, W. V.

W. V. Holt, F. Shenfield, T. Leonard, T. D. Hartman, R. D. North, and H. D. Moore, “The value of sperm swimming speed measurements in assessing the fertility of human frozen semen,” Hum. Reprod. 4(3), 292–297 (1989).
[Crossref] [PubMed]

Huser, T.

R. Heintzmann and T. Huser, “Super-Resolution Structured Illumination Microscopy,” Chem. Rev. 117(23), 13890–13908 (2017).
[Crossref] [PubMed]

Inci, F.

W. Asghar, H. Shafiee, V. Velasco, V. R. Sah, S. Guo, R. El Assal, F. Inci, A. Rajagopalan, M. Jahangir, R. M. Anchan, G. L. Mutter, M. Ozkan, C. S. Ozkan, and U. Demirci, “Toxicology Study of Single-walled Carbon Nanotubes and Reduced Graphene Oxide in Human Sperm,” Sci. Rep. 6(1), 30270 (2016).
[Crossref] [PubMed]

Isik, A.

S. Ozkavukcu, E. Erdemli, A. Isik, D. Oztuna, and S. Karahuseyinoglu, “Effects of cryopreservation on sperm parameters and ultrastructural morphology of human spermatozoa,” J. Assist. Reprod. Genet. 25(8), 403–411 (2008).
[Crossref] [PubMed]

Jahangir, M.

W. Asghar, H. Shafiee, V. Velasco, V. R. Sah, S. Guo, R. El Assal, F. Inci, A. Rajagopalan, M. Jahangir, R. M. Anchan, G. L. Mutter, M. Ozkan, C. S. Ozkan, and U. Demirci, “Toxicology Study of Single-walled Carbon Nanotubes and Reduced Graphene Oxide in Human Sperm,” Sci. Rep. 6(1), 30270 (2016).
[Crossref] [PubMed]

Joyce, G.

G. Joyce, B. D. Robertson, and K. J. Williams, “A modified agar pad method for mycobacterial live-cell imaging,” BMC Res. Notes 4(1), 73 (2011).
[Crossref] [PubMed]

Karahuseyinoglu, S.

S. Ozkavukcu, E. Erdemli, A. Isik, D. Oztuna, and S. Karahuseyinoglu, “Effects of cryopreservation on sperm parameters and ultrastructural morphology of human spermatozoa,” J. Assist. Reprod. Genet. 25(8), 403–411 (2008).
[Crossref] [PubMed]

Kaynig, V.

J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, J.-Y. Tinevez, D. J. White, V. Hartenstein, K. Eliceiri, P. Tomancak, and A. Cardona, “Fiji: an open-source platform for biological-image analysis,” Nat. Methods 9(7), 676–682 (2012).
[Crossref] [PubMed]

Kumar, N.

N. Kumar and A. K. Singh, “Trends of male factor infertility, an important cause of infertility: A review of literature,” J. Hum. Reprod. Sci. 8(4), 191–196 (2015).
[Crossref] [PubMed]

Lacy, D.

D. Lacy, A. J. Pettitt, J. M. Pettitt, and B. S. Martin, “Application of scanning electron microscopy to semen analysis of the sub-fertile man utilising data obtained by transmission electron microscopy as an aid to interpretation,” Micron (1969) 5(2), 135–173 (1974).
[Crossref]

Lansac, J.

C. Barthelemy, D. Royere, S. Hammahah, C. Lebos, M. J. Tharanne, and J. Lansac, “Ultrastructural changes in membranes and acrosome of human sperm during cryopreservation,” Arch. Androl. 25(1), 29–40 (1990).
[Crossref] [PubMed]

Lebos, C.

C. Barthelemy, D. Royere, S. Hammahah, C. Lebos, M. J. Tharanne, and J. Lansac, “Ultrastructural changes in membranes and acrosome of human sperm during cryopreservation,” Arch. Androl. 25(1), 29–40 (1990).
[Crossref] [PubMed]

Lenzi, A.

F. Pelliccione, A. Micillo, G. Cordeschi, A. D’Angeli, S. Necozione, L. Gandini, A. Lenzi, F. Francavilla, and S. Francavilla, “Altered ultrastructure of mitochondrial membranes is strongly associated with unexplained asthenozoospermia,” Fertil. Steril. 95(2), 641–646 (2011).
[Crossref] [PubMed]

Leonard, T.

W. V. Holt, F. Shenfield, T. Leonard, T. D. Hartman, R. D. North, and H. D. Moore, “The value of sperm swimming speed measurements in assessing the fertility of human frozen semen,” Hum. Reprod. 4(3), 292–297 (1989).
[Crossref] [PubMed]

Longair, M.

J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, J.-Y. Tinevez, D. J. White, V. Hartenstein, K. Eliceiri, P. Tomancak, and A. Cardona, “Fiji: an open-source platform for biological-image analysis,” Nat. Methods 9(7), 676–682 (2012).
[Crossref] [PubMed]

Martin, B. S.

D. Lacy, A. J. Pettitt, J. M. Pettitt, and B. S. Martin, “Application of scanning electron microscopy to semen analysis of the sub-fertile man utilising data obtained by transmission electron microscopy as an aid to interpretation,” Micron (1969) 5(2), 135–173 (1974).
[Crossref]

Micillo, A.

F. Pelliccione, A. Micillo, G. Cordeschi, A. D’Angeli, S. Necozione, L. Gandini, A. Lenzi, F. Francavilla, and S. Francavilla, “Altered ultrastructure of mitochondrial membranes is strongly associated with unexplained asthenozoospermia,” Fertil. Steril. 95(2), 641–646 (2011).
[Crossref] [PubMed]

Moore, H. D.

W. V. Holt, F. Shenfield, T. Leonard, T. D. Hartman, R. D. North, and H. D. Moore, “The value of sperm swimming speed measurements in assessing the fertility of human frozen semen,” Hum. Reprod. 4(3), 292–297 (1989).
[Crossref] [PubMed]

Moretti, E.

B. Baccetti, S. Capitani, G. Collodel, G. Di Cairano, L. Gambera, E. Moretti, and P. Piomboni, “Genetic sperm defects and consanguinity,” Hum. Reprod. 16(7), 1365–1371 (2001).
[Crossref] [PubMed]

E. Moretti and G. Collodel, Electron Microscopy in the Study of Human Sperm Pathologies (2012).

Mukherjee, A.

N. Naha, R. B. Bhar, A. Mukherjee, and A. R. Chowdhury, “Structural alteration of spermatozoa in the persons employed in lead acid battery factory,” Indian J. Physiol. Pharmacol. 49(2), 153–162 (2005).
[PubMed]

Mutter, G. L.

W. Asghar, H. Shafiee, V. Velasco, V. R. Sah, S. Guo, R. El Assal, F. Inci, A. Rajagopalan, M. Jahangir, R. M. Anchan, G. L. Mutter, M. Ozkan, C. S. Ozkan, and U. Demirci, “Toxicology Study of Single-walled Carbon Nanotubes and Reduced Graphene Oxide in Human Sperm,” Sci. Rep. 6(1), 30270 (2016).
[Crossref] [PubMed]

Naha, N.

N. Naha, R. B. Bhar, A. Mukherjee, and A. R. Chowdhury, “Structural alteration of spermatozoa in the persons employed in lead acid battery factory,” Indian J. Physiol. Pharmacol. 49(2), 153–162 (2005).
[PubMed]

Necozione, S.

F. Pelliccione, A. Micillo, G. Cordeschi, A. D’Angeli, S. Necozione, L. Gandini, A. Lenzi, F. Francavilla, and S. Francavilla, “Altered ultrastructure of mitochondrial membranes is strongly associated with unexplained asthenozoospermia,” Fertil. Steril. 95(2), 641–646 (2011).
[Crossref] [PubMed]

North, R. D.

W. V. Holt, F. Shenfield, T. Leonard, T. D. Hartman, R. D. North, and H. D. Moore, “The value of sperm swimming speed measurements in assessing the fertility of human frozen semen,” Hum. Reprod. 4(3), 292–297 (1989).
[Crossref] [PubMed]

Olmedo, S. B.

V. Y. Rawe, G. D. Galaverna, A. A. Acosta, S. B. Olmedo, and H. E. Chemes, “Incidence of tail structure distortions associated with dysplasia of the fibrous sheath in human spermatozoa,” Hum. Reprod. 16(5), 879–886 (2001).
[Crossref] [PubMed]

Ozkan, C. S.

W. Asghar, H. Shafiee, V. Velasco, V. R. Sah, S. Guo, R. El Assal, F. Inci, A. Rajagopalan, M. Jahangir, R. M. Anchan, G. L. Mutter, M. Ozkan, C. S. Ozkan, and U. Demirci, “Toxicology Study of Single-walled Carbon Nanotubes and Reduced Graphene Oxide in Human Sperm,” Sci. Rep. 6(1), 30270 (2016).
[Crossref] [PubMed]

Ozkan, M.

W. Asghar, H. Shafiee, V. Velasco, V. R. Sah, S. Guo, R. El Assal, F. Inci, A. Rajagopalan, M. Jahangir, R. M. Anchan, G. L. Mutter, M. Ozkan, C. S. Ozkan, and U. Demirci, “Toxicology Study of Single-walled Carbon Nanotubes and Reduced Graphene Oxide in Human Sperm,” Sci. Rep. 6(1), 30270 (2016).
[Crossref] [PubMed]

Ozkavukcu, S.

S. Ozkavukcu, E. Erdemli, A. Isik, D. Oztuna, and S. Karahuseyinoglu, “Effects of cryopreservation on sperm parameters and ultrastructural morphology of human spermatozoa,” J. Assist. Reprod. Genet. 25(8), 403–411 (2008).
[Crossref] [PubMed]

Oztuna, D.

S. Ozkavukcu, E. Erdemli, A. Isik, D. Oztuna, and S. Karahuseyinoglu, “Effects of cryopreservation on sperm parameters and ultrastructural morphology of human spermatozoa,” J. Assist. Reprod. Genet. 25(8), 403–411 (2008).
[Crossref] [PubMed]

Pelliccione, F.

F. Pelliccione, A. Micillo, G. Cordeschi, A. D’Angeli, S. Necozione, L. Gandini, A. Lenzi, F. Francavilla, and S. Francavilla, “Altered ultrastructure of mitochondrial membranes is strongly associated with unexplained asthenozoospermia,” Fertil. Steril. 95(2), 641–646 (2011).
[Crossref] [PubMed]

Pettitt, A. J.

D. Lacy, A. J. Pettitt, J. M. Pettitt, and B. S. Martin, “Application of scanning electron microscopy to semen analysis of the sub-fertile man utilising data obtained by transmission electron microscopy as an aid to interpretation,” Micron (1969) 5(2), 135–173 (1974).
[Crossref]

Pettitt, J. M.

D. Lacy, A. J. Pettitt, J. M. Pettitt, and B. S. Martin, “Application of scanning electron microscopy to semen analysis of the sub-fertile man utilising data obtained by transmission electron microscopy as an aid to interpretation,” Micron (1969) 5(2), 135–173 (1974).
[Crossref]

Pietzsch, T.

J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, J.-Y. Tinevez, D. J. White, V. Hartenstein, K. Eliceiri, P. Tomancak, and A. Cardona, “Fiji: an open-source platform for biological-image analysis,” Nat. Methods 9(7), 676–682 (2012).
[Crossref] [PubMed]

Piomboni, P.

B. Baccetti, S. Capitani, G. Collodel, G. Di Cairano, L. Gambera, E. Moretti, and P. Piomboni, “Genetic sperm defects and consanguinity,” Hum. Reprod. 16(7), 1365–1371 (2001).
[Crossref] [PubMed]

Preibisch, S.

J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, J.-Y. Tinevez, D. J. White, V. Hartenstein, K. Eliceiri, P. Tomancak, and A. Cardona, “Fiji: an open-source platform for biological-image analysis,” Nat. Methods 9(7), 676–682 (2012).
[Crossref] [PubMed]

Rajagopalan, A.

W. Asghar, H. Shafiee, V. Velasco, V. R. Sah, S. Guo, R. El Assal, F. Inci, A. Rajagopalan, M. Jahangir, R. M. Anchan, G. L. Mutter, M. Ozkan, C. S. Ozkan, and U. Demirci, “Toxicology Study of Single-walled Carbon Nanotubes and Reduced Graphene Oxide in Human Sperm,” Sci. Rep. 6(1), 30270 (2016).
[Crossref] [PubMed]

Rawe, V. Y.

V. Y. Rawe, G. D. Galaverna, A. A. Acosta, S. B. Olmedo, and H. E. Chemes, “Incidence of tail structure distortions associated with dysplasia of the fibrous sheath in human spermatozoa,” Hum. Reprod. 16(5), 879–886 (2001).
[Crossref] [PubMed]

Rawe, Y. V.

E. H. Chemes and Y. V. Rawe, “Sperm pathology: a step beyond descriptive morphology. Origin, characterization and fertility potential of abnormal sperm phenotypes in infertile men,” Hum. Reprod. Update 9(5), 405–428 (2003).
[Crossref] [PubMed]

Robertson, B. D.

G. Joyce, B. D. Robertson, and K. J. Williams, “A modified agar pad method for mycobacterial live-cell imaging,” BMC Res. Notes 4(1), 73 (2011).
[Crossref] [PubMed]

Royere, D.

C. Barthelemy, D. Royere, S. Hammahah, C. Lebos, M. J. Tharanne, and J. Lansac, “Ultrastructural changes in membranes and acrosome of human sperm during cryopreservation,” Arch. Androl. 25(1), 29–40 (1990).
[Crossref] [PubMed]

Rueden, C.

J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, J.-Y. Tinevez, D. J. White, V. Hartenstein, K. Eliceiri, P. Tomancak, and A. Cardona, “Fiji: an open-source platform for biological-image analysis,” Nat. Methods 9(7), 676–682 (2012).
[Crossref] [PubMed]

Saalfeld, S.

J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, J.-Y. Tinevez, D. J. White, V. Hartenstein, K. Eliceiri, P. Tomancak, and A. Cardona, “Fiji: an open-source platform for biological-image analysis,” Nat. Methods 9(7), 676–682 (2012).
[Crossref] [PubMed]

Sah, V. R.

W. Asghar, H. Shafiee, V. Velasco, V. R. Sah, S. Guo, R. El Assal, F. Inci, A. Rajagopalan, M. Jahangir, R. M. Anchan, G. L. Mutter, M. Ozkan, C. S. Ozkan, and U. Demirci, “Toxicology Study of Single-walled Carbon Nanotubes and Reduced Graphene Oxide in Human Sperm,” Sci. Rep. 6(1), 30270 (2016).
[Crossref] [PubMed]

Schindelin, J.

J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, J.-Y. Tinevez, D. J. White, V. Hartenstein, K. Eliceiri, P. Tomancak, and A. Cardona, “Fiji: an open-source platform for biological-image analysis,” Nat. Methods 9(7), 676–682 (2012).
[Crossref] [PubMed]

Schmid, B.

J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, J.-Y. Tinevez, D. J. White, V. Hartenstein, K. Eliceiri, P. Tomancak, and A. Cardona, “Fiji: an open-source platform for biological-image analysis,” Nat. Methods 9(7), 676–682 (2012).
[Crossref] [PubMed]

Shafiee, H.

W. Asghar, H. Shafiee, V. Velasco, V. R. Sah, S. Guo, R. El Assal, F. Inci, A. Rajagopalan, M. Jahangir, R. M. Anchan, G. L. Mutter, M. Ozkan, C. S. Ozkan, and U. Demirci, “Toxicology Study of Single-walled Carbon Nanotubes and Reduced Graphene Oxide in Human Sperm,” Sci. Rep. 6(1), 30270 (2016).
[Crossref] [PubMed]

Shenfield, F.

W. V. Holt, F. Shenfield, T. Leonard, T. D. Hartman, R. D. North, and H. D. Moore, “The value of sperm swimming speed measurements in assessing the fertility of human frozen semen,” Hum. Reprod. 4(3), 292–297 (1989).
[Crossref] [PubMed]

Singh, A. K.

N. Kumar and A. K. Singh, “Trends of male factor infertility, an important cause of infertility: A review of literature,” J. Hum. Reprod. Sci. 8(4), 191–196 (2015).
[Crossref] [PubMed]

Smith, N. I.

A. J. Hobro and N. I. Smith, “An evaluation of fixation methods: Spatial and compositional cellular changes observed by Raman imaging,” Vib. Spectrosc. 91, 31–45 (2017).
[Crossref]

Tharanne, M. J.

C. Barthelemy, D. Royere, S. Hammahah, C. Lebos, M. J. Tharanne, and J. Lansac, “Ultrastructural changes in membranes and acrosome of human sperm during cryopreservation,” Arch. Androl. 25(1), 29–40 (1990).
[Crossref] [PubMed]

Tinevez, J.-Y.

J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, J.-Y. Tinevez, D. J. White, V. Hartenstein, K. Eliceiri, P. Tomancak, and A. Cardona, “Fiji: an open-source platform for biological-image analysis,” Nat. Methods 9(7), 676–682 (2012).
[Crossref] [PubMed]

Tomancak, P.

J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, J.-Y. Tinevez, D. J. White, V. Hartenstein, K. Eliceiri, P. Tomancak, and A. Cardona, “Fiji: an open-source platform for biological-image analysis,” Nat. Methods 9(7), 676–682 (2012).
[Crossref] [PubMed]

Velasco, V.

W. Asghar, H. Shafiee, V. Velasco, V. R. Sah, S. Guo, R. El Assal, F. Inci, A. Rajagopalan, M. Jahangir, R. M. Anchan, G. L. Mutter, M. Ozkan, C. S. Ozkan, and U. Demirci, “Toxicology Study of Single-walled Carbon Nanotubes and Reduced Graphene Oxide in Human Sperm,” Sci. Rep. 6(1), 30270 (2016).
[Crossref] [PubMed]

White, D. J.

J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, J.-Y. Tinevez, D. J. White, V. Hartenstein, K. Eliceiri, P. Tomancak, and A. Cardona, “Fiji: an open-source platform for biological-image analysis,” Nat. Methods 9(7), 676–682 (2012).
[Crossref] [PubMed]

Williams, K. J.

G. Joyce, B. D. Robertson, and K. J. Williams, “A modified agar pad method for mycobacterial live-cell imaging,” BMC Res. Notes 4(1), 73 (2011).
[Crossref] [PubMed]

Arch. Androl. (1)

C. Barthelemy, D. Royere, S. Hammahah, C. Lebos, M. J. Tharanne, and J. Lansac, “Ultrastructural changes in membranes and acrosome of human sperm during cryopreservation,” Arch. Androl. 25(1), 29–40 (1990).
[Crossref] [PubMed]

Asian J. Androl. (1)

G. Cavallini, “Male idiopathic oligoasthenoteratozoospermia,” Asian J. Androl. 8(2), 143–157 (2006).
[Crossref] [PubMed]

BMC Res. Notes (1)

G. Joyce, B. D. Robertson, and K. J. Williams, “A modified agar pad method for mycobacterial live-cell imaging,” BMC Res. Notes 4(1), 73 (2011).
[Crossref] [PubMed]

Chem. Rev. (1)

R. Heintzmann and T. Huser, “Super-Resolution Structured Illumination Microscopy,” Chem. Rev. 117(23), 13890–13908 (2017).
[Crossref] [PubMed]

Fertil. Steril. (1)

F. Pelliccione, A. Micillo, G. Cordeschi, A. D’Angeli, S. Necozione, L. Gandini, A. Lenzi, F. Francavilla, and S. Francavilla, “Altered ultrastructure of mitochondrial membranes is strongly associated with unexplained asthenozoospermia,” Fertil. Steril. 95(2), 641–646 (2011).
[Crossref] [PubMed]

Hum. Reprod. (3)

W. V. Holt, F. Shenfield, T. Leonard, T. D. Hartman, R. D. North, and H. D. Moore, “The value of sperm swimming speed measurements in assessing the fertility of human frozen semen,” Hum. Reprod. 4(3), 292–297 (1989).
[Crossref] [PubMed]

V. Y. Rawe, G. D. Galaverna, A. A. Acosta, S. B. Olmedo, and H. E. Chemes, “Incidence of tail structure distortions associated with dysplasia of the fibrous sheath in human spermatozoa,” Hum. Reprod. 16(5), 879–886 (2001).
[Crossref] [PubMed]

B. Baccetti, S. Capitani, G. Collodel, G. Di Cairano, L. Gambera, E. Moretti, and P. Piomboni, “Genetic sperm defects and consanguinity,” Hum. Reprod. 16(7), 1365–1371 (2001).
[Crossref] [PubMed]

Hum. Reprod. Update (1)

E. H. Chemes and Y. V. Rawe, “Sperm pathology: a step beyond descriptive morphology. Origin, characterization and fertility potential of abnormal sperm phenotypes in infertile men,” Hum. Reprod. Update 9(5), 405–428 (2003).
[Crossref] [PubMed]

Indian J. Physiol. Pharmacol. (1)

N. Naha, R. B. Bhar, A. Mukherjee, and A. R. Chowdhury, “Structural alteration of spermatozoa in the persons employed in lead acid battery factory,” Indian J. Physiol. Pharmacol. 49(2), 153–162 (2005).
[PubMed]

J. Assist. Reprod. Genet. (1)

S. Ozkavukcu, E. Erdemli, A. Isik, D. Oztuna, and S. Karahuseyinoglu, “Effects of cryopreservation on sperm parameters and ultrastructural morphology of human spermatozoa,” J. Assist. Reprod. Genet. 25(8), 403–411 (2008).
[Crossref] [PubMed]

J. Cell Sci. (1)

C. G. Galbraith and J. A. Galbraith, “Super-resolution microscopy at a glance,” J. Cell Sci. 124(10), 1607–1611 (2011).
[Crossref] [PubMed]

J. Hum. Reprod. Sci. (1)

N. Kumar and A. K. Singh, “Trends of male factor infertility, an important cause of infertility: A review of literature,” J. Hum. Reprod. Sci. 8(4), 191–196 (2015).
[Crossref] [PubMed]

Micron (1969) (1)

D. Lacy, A. J. Pettitt, J. M. Pettitt, and B. S. Martin, “Application of scanning electron microscopy to semen analysis of the sub-fertile man utilising data obtained by transmission electron microscopy as an aid to interpretation,” Micron (1969) 5(2), 135–173 (1974).
[Crossref]

Nat. Methods (1)

J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, J.-Y. Tinevez, D. J. White, V. Hartenstein, K. Eliceiri, P. Tomancak, and A. Cardona, “Fiji: an open-source platform for biological-image analysis,” Nat. Methods 9(7), 676–682 (2012).
[Crossref] [PubMed]

Sci. Rep. (1)

W. Asghar, H. Shafiee, V. Velasco, V. R. Sah, S. Guo, R. El Assal, F. Inci, A. Rajagopalan, M. Jahangir, R. M. Anchan, G. L. Mutter, M. Ozkan, C. S. Ozkan, and U. Demirci, “Toxicology Study of Single-walled Carbon Nanotubes and Reduced Graphene Oxide in Human Sperm,” Sci. Rep. 6(1), 30270 (2016).
[Crossref] [PubMed]

Vib. Spectrosc. (1)

A. J. Hobro and N. I. Smith, “An evaluation of fixation methods: Spatial and compositional cellular changes observed by Raman imaging,” Vib. Spectrosc. 91, 31–45 (2017).
[Crossref]

Other (2)

J. Kiernan, Formaldehyde, Formalin, Paraformaldehyde And Glutaraldehyde: What They Are And What They Do (2000).

E. Moretti and G. Collodel, Electron Microscopy in the Study of Human Sperm Pathologies (2012).

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Figures (3)

Fig. 1
Fig. 1 Comparison of deconvolution microscopy (upper panels) and SIM (lower panels) images of living human spermatozoa for different probes. (a), (e) Plasma membrane labeled using CellMask Orange; (b), (f) Mitochondria labeled using MitoTracker Green; (c), (g) Nucleus labeled using Hoechst 34580; (d), (h) Microtubulin labeled using SiR-tubulin. The contrast and resolution enhancement are apparent for all probes, but most significant for the region containing mitochondria (panels (a), (b), (e) and (f)), but also for the centriole, indicated by arrows in panels (d) and (h). The images are single z-sections.

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Fig. 2
Fig. 2 Comparison of background signal in z-planes 2 (a), 3 (b) and 4 (c) counted from the agarose patch (top) used for immobilization. The distance between the z-slices is 125 nm. The agarose patch (with absorbed leftover dye) only causes significant background signal in the uppermost planes. The cells were labeled using CellMask Orange, MitoTracker Green and Hoechst 34580 and imaged live using SIM.

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Fig. 3
Fig. 3 Four-channel SIM image of living human spermatozoa labeled using CellMask Orange (yellow), MitoTracker Green (green), Hoechst 34580 (turquoise) and SiR-tubulin (magenta). The image is a 1 µm maximum intensity projected z-stack.

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Tables (1)

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Table 1 Labeling conditions applied for SIM imaging

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