Reaching deeper with 3D super-resolution microscopy

Alipasha Vaziri from the Howard Hughes Medical Institute’s Janelia Farm Research Campus is working on improving super-resolution microscopy by acquiring 3D images of thick biological samples. According to Vaziri, current super-resolution methods can only reach a few hundreds of a nanometer into a biological samples. His group wants to extend the PALM technique to depths of tens of microns and even up to the thickness of a cell.

To achieve deeper imaging, the researchers turned to temporal focusing (also called “two-photon light sculpting”). Temporal focusing uses the spectral properties of an optical pulse to shape its spatial distribution into a sheet. This sheet let the researchers activate only a thin layer of photoactivatable fluorescent proteins in different layers of a cell sample.

They demonstrated the technique on fixed cells with labeled mitochondria and on the membranes of living S2 Drosophila cells. By color-coding the position of emitters, the images could reveal depth information. The researchers achieved optical sectioning over many tens of microns using this technique, and they think that the technique could be useful for applications such as studying developmental biology on the molecular level or for researching the neural circuitry of drosophila.