title: 4D super-resolution microscopy with conventional fluorophores and single wavelength excitation in optically thick cells and tissues.
abstract: BACKGROUND
  Optical super-resolution imaging of fluorescently stained biological samples is rapidly becoming an important tool to investigate protein distribution at the molecular scale. It is therefore important to develop practical super-resolution methods that allow capturing the full three-dimensional nature of biological systems and also can visualize multiple protein species in the same sample.
  
  
  RESULTS
  We show that the use of a combination of conventional near-infrared dyes, such as Alexa 647, Alexa 680 and Alexa 750, all excited with a 671 nm diode laser, enables 3D multi-colour super-resolution imaging of complex biological samples. Optically thick samples, including human tissue sections, cardiac rat myocytes and densely grown neuronal cultures were imaged with lateral resolutions of ∼15 nm (std. dev.) while reducing marker cross-talk to <1%. Using astigmatism an axial resolution of ∼65 nm (std. dev.) was routinely achieved. The number of marker species that can be distinguished depends on the mean photon number of single molecule events. With the typical photon yields from Alexa 680 of ∼2000 up to 5 markers may in principle be resolved with <2% crosstalk.
  
  
  CONCLUSIONS
  Our approach is based entirely on the use of conventional, commercially available markers and requires only a single laser. It provides a very straightforward way to investigate biological samples at the nanometre scale and should help establish practical 4D super-resolution microscopy as a routine research tool in many laboratories.
authors.affiliation: Department of Physiology, Faculty of Medicine and Health Sciences, University of Auckland, Auckland, New Zealand.
authors.forename: David
authors.initials: D
authors.lastname: Baddeley
authors.forename: David
authors.initials: D
authors.lastname: Crossman
authors.forename: Sabrina
authors.initials: S
authors.lastname: Rossberger
authors.forename: Juliette E
authors.initials: JE
authors.lastname: Cheyne
authors.forename: Johanna M
authors.initials: JM
authors.lastname: Montgomery
authors.forename: Isuru D
authors.initials: ID
authors.lastname: Jayasinghe
authors.forename: Christoph
authors.initials: C
authors.lastname: Cremer
authors.forename: Mark B
authors.initials: MB
authors.lastname: Cannell
authors.forename: Christian
authors.initials: C
authors.lastname: Soeller
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000031:analogs & derivatives; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods*; D008962:Models, Theoretical; D032383:Myocytes, Cardiac / Q000166:cytology; D051381:Rats; D013388:Succinimides
chemical_list: C502599:Alexa Fluor 750; D013388:Succinimides; C067297:8-aminohexylamino cAMP; D000242:Cyclic AMP