Advanced Light Microscopy

Confocal Microscopy

Confocal microscopy allows increased optical resolution and contrast of by means of employing a spatial pinhole to block the out-of-focus light in the formation of an image. Recording multiple two-dimensional images at different depths enables the reconstruction of three-dimensional structures within an specimen. This technique is used extensively in life and material sciences.

Fluorescence correlation spectroscopy (FCS)

FCS is a correlation analysis of fluctuation of the fluorescence intensity. The analysis provides parameters of the physics under the fluctuations. One of the interesting applications of this is an analysis of the concentration fluctuations of fluorescent particles (molecules) in solution. In this application, the fluorescence emitted from a very tiny space in solution containing a small number of fluorescent particles (molecules) is observed. The fluorescence intensity is fluctuating due to Brownian motion of the particles. In other words, the number of the particles in the sub-space defined by the optical system is randomly changing around the average number. The analysis gives the average number of fluorescent particles and average diffusion time, when the particle is passing through the space. Eventually, both the concentration and size of the particle (molecule) are determined. Both parameters are important in biochemical research, biophysics, and chemistry.

Fluorescence cross-correlation spectroscopy (FCCS)

FCCS is essentially an extension of the fluorescence correlation spectroscopy (FCS) procedure by utilizing two differentially coloured molecules, instead of one. In other words, coincident green and red intensity fluctuations of distinct molecules correlate if green and red labelled particles are moving together through a predefined confocal volume. As a result, FCCS provides a highly sensitive measurement of molecular interactions independent of diffusion rate. This is an important advancement, given that diffusion rate depends only weakly on the size of the molecular complex.

Fluorescence-lifetime imaging microscopy (FLIM)

FLIM is an imaging technique for producing an image based on the differences in the exponential decay rate of the fluorescence from a fluorescent sample. It can be used as an imaging technique in confocal microscopy, two-photon excitation microscopy, and multiphoton tomography. The lifetime of the fluorophore signal, rather than its intensity, is used to create the image in FLIM. This has the advantage of minimizing the effect of photon scattering in thick layers of sample.

Fluorescence resonance energy transfer (FRET)

FRET is a mechanism describing energy transfer between two light-sensitive molecules (chromophores). A donor chromophore, initially in its electronic excited state, may transfer energy to an acceptor chromophore through nonradiative dipole–dipole coupling. The efficiency of this energy transfer is inversely proportional to the sixth power of the distance between donor and acceptor, making FRET extremely sensitive to small changes in distance. Measurements of FRET efficiency can be used to determine if two fluorophores are within a certain distance of each other. Such measurements are used as a research tool in fields including biology and chemistry.