The introduction of immunofluorescence by Coons in early 1940 and fluorescence microscopy with incident light illumination by Ploem in 1967 has set the scene for decades, in which Abbe’s law defined the achievable spatial resolution. At present, advanced microscopic techniques have allowed us to observe molecular machines at work in intact living cells at nm resolution, bypassing the diffraction limit whereas light sheath illumination (instead of epi-illumination) has become a superior alternative for many applications. Green fluorescent proteins have replaced good old fluorescein and rhodamine dyes, and optogenetic tools allow us to introduce function with light. Microscope systems have become “filter less” with the introduction of optoacoustic devices and “white lasers” are used as excitation light sources. This presentation will look back at 50 years of advanced microscopy and show some of the highlights in the field of automated cell analysis, chromosome analysis by FISH karyotyping, and rare event detection.
How is advanced microscopy positioned in an “omics world”? The overwhelming amount of information that has become available after sequencing the human genome, and subsequent developments in “omics-type” of disciplines, have learned us a lot about the molecular composition of cells. How regulatory mechanisms form the basis of cell growth, differentiation and death in normal tissue, but also in case of disease, is not yet clear in many cases. Notably, a protein can only exercise its function if it is present at the right time, in the right amount, properly activated and in the presence of other proteins, at a particular position in the cells. Thus, spatio-temporal information is crucial which is not provided by “omics technologies” only.
In this context combined use of light- and high resolution microscopy (CLEM) as well as cryo-tomography and 3D EM is a powerful combination. For instance, for the study of the production of Von Willebrand factor and Weibel Palade bodies (1), the barrier function of the glomerular endothelial surface layer in the kidney and the growth and differentiation of Streptomyces, the main commercial producing micro-organism of antibiotics. Also, a technique will be presented to generate and visualise ultra-large high resolution microscopy maps (2).
[1] M.J. Mourik. J.A. Valentijn, J. Voorberg, A.J. Koster, K.M. Valentijn and J. Eikenboom. “Von Willebrand factor remodeling during exocytosis from vascular endothelial cells”. J. Tromb Haemost. 11, 2009-2011 (2013).
[2] F.G.A. Faas, M.C. Avramut,1 B.M. van den Berg, A.M. Mommaas, A.J. Koster and R.B.G. Ravelli. “ Virtual nanoscopy: Generation of ultra-large high resolution electron microscopy maps”. J Cell Biol 198, 457-469 (2012).
