How The Diffraction Barrier Was Broken to Achieve Sub 80 nm Optical Microscopy

A recent article in BioTechniques tells the story of how the diffraction barrier, that’s been long thought to fundamentally limit the possible resolution of far-field fluorescence microscopy, was overcome with fresh scientific thinking and solid engineering.

It starts with initial theoretical work of physicist Stefan Hell and how it led to the technology that Leica later licensed and proceeded to commercialize into a real microscope.

Hell, though, had a gut feeling the barrier was less solid than imagined. He suspected it might be possible to overcome it by manipulating the “light-driven state transitions” of fluorescent dyes: in other words tinkering with the process by which the dyes absorb and release energy. He pursued the idea in the early 1990s, first at the European Molecular Biology Laboratory and then at the University of Turku in Finland, where he had a senior postdoctoral fellowship. In 1993, he had a breakthrough. “I realized that one could do it by turning off a dye by stimulated emission.”

Hell’s vision was to use two superimposed laser spots, an excitation beam and a beam for molecularde-excitation, and alternate their pulses so that some of the molecules in the excitation area are prevented from fluorescing – the foundation of STED. “STED introduces a mechanism by which we keep molecules dark even though they are illuminated with excitation light,” he says.

As in standard confocal microscopy, the excitation beam excites the fluorophores in a diffraction-limited region in the sample, rasterizing across the sample and collecting fluorescence intensity spot-by-spot. First, though, the STED beam—shaped like a doughnut with a hole in the center—deactivates the dyes at the periphery of that relatively large spot by forcing them back down to their ground state without fluorescing, acting like a light-based photomask. The net result is to shrink the effective excitation region below the diffraction limit.

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