LN2 – Light Nanoscopy : Second Generation – New approaches for super-resolution microscopy

In recent years, our laboratory “Bioenergetics, Metalloproteins and Stress” has developed a patented method for obtaining super-resolution fluorescence images of biological samples, based on a classical optical approach. We are able to obtain images at super-resolution at a price considerably cheaper than the one indicated by commercial machines.

Our method is based on the use of nanodetectors, from which we may, in a first step, extract an image at a resolution nearly twice as high as classical confocal scanning microscopes. Then, in a second step, we may perform a global analysis of all the detectors used, to obtain a resolution close to STED systems, if not higher (experiments still ongoing).

A drawback of our method is its speed, as it necessitates to scan the sample relatively slowly to reach a high resolution (typically couple of minutes to scan one bacterium). In the framework of the DIM ELICIT, we developed at Saclay an optical system to record fluorescent images at different wavelengths simultaneously during the same scan.

Images obtained from a cyanobacterium, locating a GFP-tagged protein (left), and the photosynthetic membranes (right).
Left : fluorescence at 530 n ;,Right : fluorescence at 660 nm. Excitation 488 nm.

This method circumvents all the pitfalls bound to successive scannings (image shifts, evolution of the sample) and can be generalized to four detection wavelengths. It was successfully applied to E. coli cells, stained both to reveal lipids and to reveal DNA (specifically prepared in the laboratory of Oliver Espeli – College de France). The resulting images allow us to position the nucleoid in the cells in a better way, thus opening the way for high-resolution structural analysis of the bacterial genome during cell cycle.