Immunofluorescence

Immunofluorescence is an technique which uses the highly specific binding of an antibody to its antigen in order to label specific proteins or other molecules within the cell. A sample is treated with a primary antibody specific for the molecule of interest. A fluorophore can be directly conjugated to the primary antibody. Alternatively a secondary antibody, conjugated to a fluorophore, which binds specifically to the first antibody can be used. For example a primary antibody raised in a mouse which recognises tubulin combined with a secondary anti-mouse antibody derivatised with a fluorophore could be used to label microtubules in a cell.

Fluorescent proteins

The modern understanding of genetics and the techniques available for modifying DNA allows scientists to genetically modify proteins to also carry a fluorescent protein reporter. In biological samples this allows a scientist to directly make a protein of interest fluorescent. The protein location can then be directly tracked, including in live cells.

Limitations

Fluorophores lose their ability to fluoresce as they are illuminated in a process called photobleaching. Photobleaching occurs as the fluorescent molecules accumulate chemical damage from the electrons excited during fluorescence. Photobleaching can severely limit the time over which a sample can be observed by fluorescent microscopy. Several techniques exist to reduce photobleaching such as the use of more robust fluorophores, by minimizing illumination, or by using photoprotective scavenger chemicals.

Fluorescence microscopy with fluorescent reporter proteins has enabled analysis of live cells by fluorescence microscopy, however cells are susceptible to phototoxicity, particularly with short wavelength light. Furthermore fluorescent molecules have a tendency to generate reactive chemical species when under illumination which enhances the phototoxic effect.

Unlike transmitted and reflected light microscopy techniques fluorescence microscopy only allows observation of the specific structures which have been fluorescently labeled. For example observing a tissue sample prepared with a fluorescent DNA stain by fluorescent microscopy only reveals the organisation of the DNA within the cells and reveals nothing else about the cell morphologies.

Improvements and sub-diffraction techniques

The wave nature of light limits the size of the spot to which light can be focused due to the diffraction limit. This limitation was described in the 19th century by Ernst Abbe and limits an optical microscope's resolution to approximately half of the wavelength of the light used. Fluorescence microscopy is central to many techniques which aim to reach past this limit by specialised optical configurations.

Several improvements in microscopy techniques have been invented in the 20th century and have resulted in increased resolution and contrast to some extent. However they did not overcome the diffraction limit. In 1978 first theoretical ideas have been developed to break this barrier by using a 4Pi microscope as a confocal laser scanning fluorescence microscope where the light is focused ideally from all sides to a common focus which is used to scan the object by 'point-by-point' excitation combined with 'point-by-point' detection. However, the first experimental demonstration of the 4pi microscope took place in 1994. 4Pi microscopy maximizes the amount of available focusing directions by using two opposing objective lenses or Multi-photon microscopy using redshifted light and multi-photon excitation.

The first technique to really achieve a sub-diffraction resolution was STED microscopy, proposed in 1994. This method and all techniques following the RESOLFT concept rely on a strong non-linear interaction between light and fluorescing molecules. The molecules are driven strongly between distinguishable molecular states at each specific location, so that finally light can be emitted at only a small fraction of space, hence an increased resolution.

As well in the 1990s another super resolution microscopy method based on wide field microscopy has been developed. Substantially improved size resolution of cellular nanostructures stained with a fluorescent marker was achieved by development of SPDM localization microscopy and the structured laser illumination (spatially modulated illumination, SMI). Combining the principle of SPDM with SMI resulted in the development of the Vertico SMI microscope. Single molecule detection of normal blinking fluorescent dyes like Green fluorescent protein (GFP) can be achieved by using a further development of SPDM the so-called SPDM phymod technology which makes it possible to detect and count two different fluorescent molecule types at the molecular level (this technology is referred to as 2CLM, 2 Color Localization Microscopy).

Alternatively, the advent of photoactivated localization microscopy could achieve similar results by relying on blinking or switching of single molecules, where the fraction of fluorescing molecules is very small at each time. This stochastic response of molecules on the applied light corresponds also to a highly nonlinear interaction, leading to subdiffraction resolution.