Balanced super-resolution optical fluctuation imaging
Three-dimensional fluorescence microscopy
Super-resolution optical fluctuation imaging (SOFI) achieves 3D super-resolution by computing temporal cumulants or spatio-temporal cross-cumulants of stochastically blinking fluorophores [1,2]. In contrast to localization microscopy, SOFI is compatible with weakly emitting fluorophores and a wider range of blinking conditions . Balanced SOFI analyses several cumulant orders for extracting molecular parameter maps, such as the bright and dark state lifetimes, the concentration and the brightness distributions of fluorophores within biological samples [a]. In combination with a deconvolution of the cumulant images, the estimated parameter maps proved useful to balance the image contrast and to linearize the brightness and blinking response. Thereby, the image quality and the resolution were improved significantly (see for example figure 1).
Figure 1: Alexa647-labeled alpha-tubulin in fixed HeLa cells imaged with a total internal reflection fluorescence microscope and analyzed to the 5. cumulant order [a]. Left: labeling density showing the alpha-tubulin structure. Right: on-time ratio distribution reporting variations in the excitation intensity due to the axial position of the tubuli in the evanescent excitation field. The lateral resolution is about 70nm.
This work has been funded by the Swiss National Science Foundation (SNF).
The following toolboxes are published under the GNU General Public License version 3 for free use and modification. By downloading these files you accept the copyright terms.
© 2014 École Polytechnique Fédérale de Lausanne