Microparticle characterization via confocal microscopy

A brief history of confocal microscopy

Confocal microscopy was invented by Marvin Minsky (FIGURE) in 1957, and subsequently patented in 1961. Minsky was trying to study neural networks to understand how brains learn, and needed a way to image these connections in their natural state (in three dimensions). He invented the confocal microscope in 1955, but its utility was not fully realized until technology could catch up. In 1973 Egger published the first recognizable cells, and the first commercial microscopes were produced in 1987.

In the 1990's confocal microscopy became near routine due to advances in laser technology, fiber optics, photodetectors, thin film dielectric coatings, computer processors, data storage, displays, and fluorophores. Today, confocal microscopy is widely used in life sciences to study cells and tissues.

The basics of fluorescence

Fluorescence is the emission of a secondary photon upon absorption of a photon of higher wavelength. Most molecules at normal temperatures are at the lowest energy state, the so-called 'ground state'. Occasionally, a molecule may absorb a photon and increase its energy to the excited state . From here it can very quickly transfer some of that energy to other molecules through collisions; however, if it cannot transfer enough energy it spontaneously emits a photon with a lower wavelength [link] . This is fluorescence.

In fluorescence microscopy, fluorescent molecules are designed to attach to specific parts of a sample, thus identifying them when imaged. Multiple fluorophores can be used to simultaneously identify different parts of a sample. There are two options when using multiple fluorophores:

• Fluorophores can be chosen that respond to different wavelengths of a multi-line laser.
• Fluorophores can be chosen that respond to the same excitation wavelength but emit at different wavelengths.

In order to increase the signal, more fluorophores can be attached to a sample. However, there is a limit, as high fluorophore concentrations result in them quenching each other, and too many fluorophores near the surface of the sample may absorb enough light to limit the light available to the rest of the sample. While the intensity of incident radiation can be increased, fluorophores may become saturated if the intensity is too high.

Photobleaching is another consideration in fluorescent microscopy. Fluorophores irreversibly fade when exposed to excitation light. This may be due to reaction of the molecules’ excited state with oxygen or oxygen radicals. There has been some success in limiting photobleaching by reducing the oxygen available or by using free-radical scavengers. Some fluorophores are more robust than others, so choice of fluorophore is very important. Fluorophores today are available that emit photons with wavelengths ranging 400 - 750 nm.