Fluorescence techniques are invaluable for investigating biological questions. Unfortunately, the spatial resolution of conventional fluorescence microscopy is limited by diffraction to ~250 nm. The length scales relevant to many biological processes are on the order of tens of nanometers, an order of magnitude smaller than the resolution limit. Therefore, developing novel fluorescence microscopy techniques for biological imaging at the nanometer scale is an exciting field that is growing rapidly.

Scales of Nature

STED Microscopy

The recent development of nanoscopy has broken the conventional resolution barrier and advanced fluorescence microscopy to a new imaging frontier. For example, stimulated emission depletion (STED) nanoscopy uses (in addition to a fluorescence excitation laser) a second, ring-shaped laser to quench fluorescence. By increasing the intensity of this laser, fluorescence is confined to a volume that can, in principle, be made arbitrarily small (Fig. 1). By exploiting the on/off switching properties of fluorescent molecules in a targeted manner, the long-standing resolution limited imposed by diffraction can be overcome and spatial resolution approaching the molecular level is now possible.

STED vs Confocal Fig v1

Adaptive Optics

In practice, the obtainable resolution of a fluorescence microscope can often be limited by optical aberrations. For example, imperfections in the quality of the laser focus will rapidly degrade the image resolution and signal-to-noise ratio. These adverse effects are of particular concern when imaging thick biological tissue. Fortunately, the use of an adaptive optical element such as a spatial light modulator or deformable mirror can be used to correct for sample-induced aberrations and restore image quality.

Adaptive Optics Fig v1

Fluorescence Correlation Spectroscopy

Complimentary to the improved spatial resolution provided by fluorescence nanoscopy, fluorescence correlation spectroscopy (FCS) provides access to dynamic processes over time scales ranging from seconds to nanoseconds. Over the past several decades FCS has been an imperative tool for measuring diffusion coefficients, chemical rates and concentrations, rotational kinetics, and excited-state dynamics.

FCS Figure v1