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.
Methodology/Principal Findings: 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/Significance: 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. |
