Researchers from the Max-Planck-Institut (Göttingen, Germany) claim to have broken the diffraction barrier responsible for finite focal spot size and limited resolution in far-field fluorescence microscopy by quenching excited organic molecules at the rim of the focal spot through stimulated emission. By forcing excited molecules into an upper vibrational level of the ground state, the stimulation process can prevent re-excitation by the same beam, thereby enabling the cessation of fluorescence. The Max-Planck group was successful in applying this "engineering" of the fluorescence to biological imaging in a sample of live bakers yeast and E. coli cells.
The light source for the experiment was a mode-locked Ti:sapphire laser emitting pulses in the near infrared at a repetition rate of 76 MHz. Two synchronized pulse trains were obtained (one in the visible and one in the near-infrared) using an optical parametric oscillator with an intracavity frequency doubler to convert some of the pulses into the visible. The pulse trains were subsequently adjusted in an optical delay stage and coupled into the experimental setup using dichroic mirrors. Visible 0.2-ps pulses provided excitation, while grating-stretched, 40-ps, near-infrared pulses provided stimulated emission depletion (STED). The extension of the STED pulses well beyond the roughly 0.2-ps relaxation time was the factor that ensured quenched molecules would not be re-excited by the same beam though vibrational relaxation.
An excitation wavelength of 560 nm and an STED wavelength of 765 nm were used for dyes with fluorescence wavelengths between 650 and 800 nm, and fluorescence was collected in the back-propagating mode as the stimulating light beam passed through the sample.
The researchers reported up to a factor-of-six reduction in spot size beyond the diffraction barrier along the optic axis and a factor-of-2 improvement in the radial direction, for a factor-of-18 reduction in spot volume beneath that of confocal microscopy. They added that the reduction factors could be further reduced by increasing STED intensity, while maintaining a zero STED-point-spread-function (PSF) intensity in the center and increasing it toward the periphery. Photostress on the dye and sample would ultimately limit the path to further spot size reductions based on intensity increases, however.
In addition to the experimental use with fluorescence, STED is "also applicable to nonfluorescent organic molecules with sufficient excited state lifetimes and significant stimulated emission cross-sections," the authors wrote.1 "In fact, our microscope demonstrates the control of the temporal distribution of molecules in their excited state. As the excited state is a first step in chemical transitions, our concept should enable restriction of chemical reaction, such as those occurring in photo induced, three-dimensional data storage, to a spatial volume hitherto inconceivable with focused light."
REFERENCE
- T. A. Klar et al,, Proc. Natl. Acad. Sci. USA 97(15), 8206 (July 18, 2000).