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Translation, Touch, and Overlap in Multi-fluorescence Confocal Laser Scanning Microscopy to Quantitate Synaptic Connectivity

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Laser Scanning Microscopy and Quantitative Image Analysis of Neuronal Tissue

Part of the book series: Neuromethods ((NM,volume 87))

Abstract

Imaging of cellular detail requires an imaging system that provides sufficient resolution. In a confocal laser scanning microscope (CLSM), microscope resolution (the optical component) and pixel resolution (the computer component) are extremely important issues. Microscope resolution depends on refraction inherent in the selected objective lens. The refractive properties and therefore the resolution of a lens are expressed by its numerical aperture (NA). Abbe’s equation, which includes also the wavelength of the used light, is the mathematical expression that determines optical resolution. The highest resolution is obtained using a high NA oil immersion lens where the oil has the same refractive properties as the mounting medium. As wet mounting preserves 3D better than mounting followed by drying and embedding in an organic mounting medium, a water immersion objective lens might be preferred when cultured cells are the subject of study.

Pixel size should preferably equal one half of the (radial) Abbe resolution of the optical instrument. For Nyquist sampling, the smallest feature should be at least 4 pixels wide (“Pawley’s Four”). Structures should at least be 10 pixels across to do meaningful 3D reconstruction. Image deconvolution is recommended. Colocalization of signal in a multi-fluorochrome stained specimen consists of statistical overlap of populations of pixels measured in different channels in the CLSM. Images of small biological objects that are in physical contact (“touch”) show always a small footprint area wherein imaging signals overlap. Controls are vital.

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Acknowledgements

We thank Amber Boeve-Boekel, Danielle Versendaal, Verena Aliane, Irineu Bochaca, Robert Schuit, Jean-Luc Boulland, and Tim van Groningen for their ever-enthusiastic confocal imaging efforts and the discussions about the translational process from biological object, via laser scanning imaging, to aggregates of high-intensity voxels in a voxel matrix.

Author information

Authors and Affiliations

  1. Department of Anatomy and Neurosciences, VU University Medical Center, Amsterdam, The Netherlands

    Floris G. Wouterlood

  2. Department of Pathology, VU University Medical Center, Amsterdam, The Netherlands

    Jeroen A. M. Beliën

Authors
  1. Floris G. Wouterlood
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  2. Jeroen A. M. Beliën
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Editor information

Editors and Affiliations

  1. Department of Neurobiology, University of Osnabrück, Osnabrück, Germany

    Lidia Bakota

  2. Department of Neurobiology, University of Osnabrück, Osnabrück, Germany

    Roland Brandt

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Wouterlood, F.G., Beliën, J.A.M. (2014). Translation, Touch, and Overlap in Multi-fluorescence Confocal Laser Scanning Microscopy to Quantitate Synaptic Connectivity. In: Bakota, L., Brandt, R. (eds) Laser Scanning Microscopy and Quantitative Image Analysis of Neuronal Tissue. Neuromethods, vol 87. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0381-8_1

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  • DOI: https://doi.org/10.1007/978-1-4939-0381-8_1

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-0380-1

  • Online ISBN: 978-1-4939-0381-8

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