BioEssays

Light Microscopy: From imaging probes to high resolution techniques andcomputational image analysis. On pages 333–340 Jason Swedlow gives a nice overview over the topics covered in this Special Issue. Advances in optics, automation, computational tools and imaging probes allow the visualization of ever smaller structures at increasing resolution. Furthermore, these techniques can be used for a wide variety of applications from basic research to clinical domains, e.g. by employing imaging techniques for screening.

The image shows a section of mouse small intestine stained with DAPI (blue), anti-tubulin (red) and phalloidin (green).

Recent advances in biological microscopy (such as FLIM, FCS, PALM, STED) have also led to new applications, from basic research to preclinical and even clinical domains. Furthermore, these methods generate huge and complex datasets and thus require increasingly sophisticated downstream processing and analysis.

Different methods can be used to improve FRET, such as spectral bleedthrough correction and acceptor photobleaching methods (for the quantification of FRET signals) or spectral imaging and FLIM (fluorescence lifetime imaging microscopy). The discovery of new fluorescent proteins (e.g. from corals) will broaden the possibilities to explore new FRET pairs.

SHG nanoprobes are nanoparticles (e.g. crystals such as barium titanate BaTiO₃) that produce light through a mechanism known as second harmonic generation (SHG). Because of their photophysical properties they offer unique advantages for molecular imaging studies, especially in single molecule and deep-tissue imaging.

Fluorescence correlation spectroscopy (FCS) allows the measurement of concentrations, mobilities, and interactions of fluorescent biomolecules based on the statistical analysis of intensity fluctuations. It has already been applied to a wide range of research topics, such as the study of single molecule translocations through nuclear pores or the determination of the stoichiometry of molecular complexes at adhesions.

Depending on the application, different FRET methods can be used: the ratio approach is useful for detecting intramolecular FRET, while fluorescence lifetime imaging microscopy (FLIM) should be used for quantitatively detecting intermolecular interactions.

Recently, different fluctuation correlation methods, such as fluorescent correlation spectroscopy (FCS), raster image correlation spectroscopy (RICS) and pair correlation function have been developed. These methods can be used to examine various kinds of dynamics: protein diffusion and interaction, protein aggregation, detection of barriers to diffusion etc.

Stimulated emission depletion (STED) microscopy allows researchers to overcome the diffraction barrier, and has the advantage that the high-resolution image is obtained in a direct fashion. Such super-resolution imaging revealsgreaterbiological complexity than hitherto witnessed. It increasingly necessitates new interpretations of data, and sometimes revises our understanding ofbiological structures and mechanisms.

Inphotoactivated localization microscopy (PALM) photoactivable fluorescent proteins (PA-FPs) are stochastically activated, imaged, their localization determined and then bleached. By repeating this cycle with different subsets of PA-FPs and combining the data, a super-resolution image is obtained. An overview of latest developments in pair-correlation analysis is given here.

Light sheet microscopy is ideally suited to in vivo imaging over long periods of time as it not only offers low photo-toxicity but also high image acquisition rates. One example of this technique is selective plane illumination microscopy (SPIM) which, for example, even allows imaging of entire embryos with isotropic resolution.

3D-structured illumination microscopy (3D-SIM) can be used to study nuclear architecture at the ultrastructural level.This method can also be combined with 3D fluorescence in situ hybridization (3D-FISH) for the topographical analysis of defined nuclear targets. The proof of principle of this combination of methods lies in the high degree of structural preservation of chromatin presented here.

Modern microscopy techniques generate large amounts of data that require subsequent computational image processing, analysis and modeling. These image analysis tools include methods to reduce noise and enhance features of fluorescence images in order to determine cell contours and to precisely localize fluorescent spots.