Introduction to light microscopy: from the basics to advanced and beyond.
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1
VIB/KU Leuven, VIB Center for the Biology of Disease, KU Leuven, Belgium
Light microscopy is holding a special place in life science, starting with proving the very existence of cells. Since then microscopy has allowed for new insights into the mysteries of life like no other technique. In contrast to other techniques, microscopy offers direct readout of the information on different levels. Like this the light microscope has become a truly indispensable part of instrumentation for the life science laboratory. The level of sophistication for microscopes varies to a large extent ranging from devices that provide illumination of the sample and the magnification of the resulting optical image to complex automated machines that require intricate sample preparation and expert knowledge to operate.
In the simplest case the microscope provides a basic 2D image. However developments of the last decades made new dimensions accessible for the researcher. Confocal microscopy allows not only rejecting out of focus light, it as well made the third spatial dimension available and is thus rendering the 2D pixel information into a 3D volumetric – voxel information. In addition time and spectral information are adding more dimensions that can be investigated (Pawley, 2006).
Currently we see how the technology is pushed to provide ever increasing resolution in space and time (Galbraith and Galbraith, 2011; Munck et al., 2012).
In addition to multidimensional image acquisition, light microscopy simultaneously captures information on the morphological, subcellular, molecular, organ and organism level. At the same time different physical properties of the sample can easily be interrogated. These physical properties that can be read out are ranging from quantifiable intensity over polarization to fluorescence lifetime, anisotropy, concentration and many other properties (Pawley, 2006).
The recent past has seen an advent of new microscopy techniques published. The degree of sophistication and specialization leading to new and different devices has reached an all-time peak. Moreover microscopy proves to be a truly interdisciplinary topic with new methods being the results of the cross talk between developers and application specialists from different areas across all disciplines from mathematics and physics to chemistry and biology (Pawley, 2006).
In this introductory lecture, we will touch upon different techniques and we will outline contrast methods, fluorescence microscopy, confocal setups, 3D imaging, and the fundamentals of resolution. We will highlight novel methods for sub-diffraction limit imaging and other advanced light microscopy techniques.
References
Pawley, J. (2006). Handbook of Biological Confocal Microscopy. New York Springer.
Galbraith C.G., Galbraith J.A. (2011). Super-resolution microscopy at a glance. J. Cell Sci. 124, 1607-1611.
Munck S., Miskiewicz K., Sannerud R., Menchon S.A., Jose L., Heintzmann R., Verstreken P., and Annaert W. (2012) Sub-diffraction imaging on standard microscopes through photobleaching microscopy with non-linear processing. J. Cell Sci. 125, 2257-2266.
Keywords:
image acquisition,
Microscopy, Fluorescence,
contrast,
super-resolution microscopy,
imaging,
biomarkers
Conference:
Imaging the brain at different scales: How to integrate multi-scale structural information?, Antwerp, Belgium, 2 Sep - 6 Sep, 2013.
Presentation Type:
Lecture
Topic:
Image acquisition across different modalities
Citation:
Munck
SL
(2013). Introduction to light microscopy: from the basics to advanced and beyond..
Front. Neuroinform.
Conference Abstract:
Imaging the brain at different scales: How to integrate multi-scale structural information?.
doi: 10.3389/conf.fninf.2013.10.00015
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Received:
30 Jun 2013;
Published Online:
30 Aug 2013.
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Correspondence:
Dr. Sebastian L Munck, VIB/KU Leuven, VIB Center for the Biology of Disease, KU Leuven, Leuven, 3000, Belgium, sebastian.munck@cme.vib-kuleuven.be