Subdiffraction Microscopy

In the life sciences, structure and function are very closely linked. This is why you have to know about the first in order to understand the second. Here, light microscopes are an essential tool because they yield easily interpretable structural information quickly. 

In order to get a high contrast, fluorescence is typically used. That is, the structures to be observed are tagged with molecules that emit light with a characteristic spectrum (so called fluorophores) and only the light in this spectrum is recorded. However, resolution is intrinsically limited by diffraction.

Other microscopy techniques like electron microscopy have a much higher resolution but suffer from severe disadvantages. For example, the sample has to be prepared in a way that changes its properties drastically.

In the search for a microscopy technique with a biocompatible sample environment in combination with a high resolution, people have come up with the idea to use nonlinear properties of fluorophores in several ways.

We use a technique called stimulated emission depletion (STED, see www.nanoscopy.de), with which fluorophores are “turned off” by light so that only a subdiffraction-sized region of fluorophores remains active at a time.

Working Principle

The image of a point source in an optical system is called the point spread function (PSF). Because no system can transmit all fourier components into the image, the PSF cannot become infinitely narrow. In a usual setup, the form of the PSF is given by the Airy function A(x) = J_1(x)/x, where J_1(x) is the first order bessel function of the first kind. Due to this, two point sources can only be resolved if they are far enough apart to separate the corresponding Airy patterns.

This minimal resolvable distance is d_min=lambda/(2NA), where NA=n sin(alpha) is the numerical aperture and alpha is the half-aperture angle of the objective.

 

In STED, we squeeze the PSF by making use of stimulated emission. This term describes the fact that excited fluorophores can be deexcited by light with a wavelength the emission spectrum. Upon excitation of a usual, Bessel-shaped region, deexciting light having zero intensity where the excitation light is maximal is shone on the sample. In this way, the outer region is deexcited, yielding less signal. Thereby the effective PSF is squeezed and the minimal resolvable distance gets smaller.