Optical excitation at the microscopic scale is used in a large variety of domains such as : the generation of the fluorescence of biological markers or the photochemistry in resins for the microlithography which are fundamental techniques in biology-medicine and in microelectronics. However, the use of radiation in the visible range, with excitation or emission wavelengths in the micrometer range, limits the achievable spatial resolution to about 200 nm (due to diffraction laws), which is more than 10 times the size of macromolecular complexes. Indeed microlithography as well cannot accept this limitation.
Recently, significant efforts have been devoted to overcoming this resolution limit in fluorescence microscopy, leading to so-called “super resolution” or “nanoscopy” methods. The 2014 Nobel prize in chemistry recognizes these efforts . Among them, STED microscopy (for Stimulated Emission Depletion) is one the most promising.
STED It is based on the control of fluorophores excited state population by stimulated emission. Two laser beams are used : the first to excite the molecule and a second to deplete the excited state and force the molecule back to the fundamental state where it cannot emit fluorescence (or generate a photochemical reaction). If the second beam is doughnut-shaped with a central zero, fluorescence emission will be inhibited everywhere except at the very center, thus enhancing the spatial resolution.
Although this technique has demonstrated a resolution down to 20 nm, its wide-spread use is presently limited by the complexity and cost of a STED setup. In a standard STED setup, two different wavelengths, usually delivered by distinct laser sources, need to be synchronized temporally, independently shaped and overlapped in the focal plane of the microscope objective.
At the Laboratoire Interdisciplinaire de Physique LIPhy, the research team " Matériaux, Optique et techniques Instrumentales pour le vivant" MOTIV is already engaged in an ambitious project devoted to the STED technique, and it has recently demonstrated its ability to innovate by developing an original methods.
Through the present project we now plan to develop a more practical apparatus : a STED microscope which uses a micro-chip laser that delivers simultaneously two wavelengths. This laser is provided by the Grenoble laser company TEEM-PHOTONICS. The wavelengths 355 nm and 532 nm are obtained by harmonic conversion from a Nd-YAG laser emission. This source should allow a simplified STED scheme since the two beams (355 nm for excitation and 532 nm for depletion) are intrinsically aligned and synchronized. This new concept will be more efficient than the commercial system since it will take advantage of the subnanosecond pulsed excitation and stimulation well adapted to the dynamics characteristics of the most popular fluorescent contrast agents. Finally the coast of a complete system based on microchip lasers will be strongly reduced since their prices are much lower than those used in the commercial STED microscope.
In this NANOTECHNOLOGIES & NANOSYSTEMS project, at LIPhy, with the collaboration of the Grenoble Institut of Neurosciences GIN and the competence of the TEEM-Photonics company :
We are studying and developping a high repetition rate microchip laser,
Simultaneously we optimise the beam shaping.
Build up a prototype of microscope consisting in a combination of STED and Light-sheet illumination .
A validation by imaging nanoobjects and biological tissues is in progress.