Optogenetics: Light-driven Actuators and Light-emitting Sensors in Cell Biology
CHAPTER 2: Super-resolution Microscopy
Published:18 Sep 2018
Xiyu Yi, Tal-Zvi Markus, Xavier Michalet, Shimon Weiss, David Bensimon, 2018. "Super-resolution Microscopy", Optogenetics: Light-driven Actuators and Light-emitting Sensors in Cell Biology, Sophie Vriz, Takeaki Ozawa
Download citation file:
In a regular microscope, the fluorescence of molecules is excited by light of wavelength λex focused by an objective lens characterized by its numerical aperture (NA). Light emitted by these fluorophores (at wavelength λem > λex) is collected by the same objective lens and imaged by the tube lens as a spot (the point spread function (PSF) of the microscope) of radius rAiry = 0.61λem/NA (see Figure 2.1). Two point sources separated by a distance d significantly smaller than rAiry cannot be resolved easily,1 as their PSFs merge into a single spot (the so-called Rayleigh criterion). This diffraction effect has defined the resolution limit of microscopes for more than a century and has been one of the motivations for the development of electron microscopy. According to the Rayleigh criterion, the classical resolution limit of a good microscope objective (i.e. with NA = 1.4) is 0.44λem. Thus, when observing cellular structures labeled with fluorophores such as the green fluorescent protein (GFP), emitting at λem = 510 nm, details below 200 nm cannot be resolved. In the past 10 years, methods have been developed that overcome this constraint and allow for almost unlimited resolution (i.e. super-resolution) in optical microscopy.