MBI-8003 Korrelativ lys- og elektronmikroskopi (CLEM) - 5 stp

The course is intended for PhD students who want to learn more about the modern use of electron microscopy and CLEM as research tools in medicine and biology

The course is organized by the Department of Medical Biology and the Norwegian Research School in Medical Imaging (MEDIM).

Travel grants

The Norwegian Research School in Medical Imaging (MedIm) provides travel grants for all participating PhD-students from other Norwegian universities. The grant covers travel expenses to and from Tromsø, and housing in Tromsø during the course. See this page for more information: www.medicalimaging.no

• CLEM in medical and biological research - applications, instrumentation, sampling, probes
• Methods for detection/fine localization of proteins (on section, pre embedding, and by use of in vivo expressed tags)
• How to find the needle in the haystack - techniques to add landmarks on cells and bulk specimen
• Sample preparations for CLEM - 1) the Tokuyasu technique; 2) high pressure freezing/freeze substitution, 3) microwave processing
• Ultramicrotomy of resin-embedded and frozen specimens
• Immunolabeling of sections for light and electron microscopy , and special techniques for CLEM on cells and bulk specimen
• Flat embedding techniques and preparation of culture substrates for CLEM
• Transmission electron microscopy
• Confocal microscopy
• Image analysis

Recommended prerequisites:

Applicants who are affiliated with the national network "Norwegian Research School in Medical Imagining" (MedIm), will be prioritized for admission if the number of applicants exceeds the course capacity of 12 students.

For further application details: uit.no/helsefak/forskning/phd/emner

Knowledge: Upon completion of this course the participants will be able to explain relevant modern methodologies/techniques using CLEM as research tools.

Skills: In this part the student will be able to explain:

• Different methods of preparation (including sampling, fixation, processing, immune labeling) of cells and tissues for CLEM.
• The theoretical basis for EM and confocal microscopy.
• Practical use of instruments.
• Image analysis

Competence: Basic competence to conduct experiments using CLEM as a method.

Lectures, demonstrations and practical exercises.

Guest lecturers this semester: Heinz Schwarz, Yannick Schwab, Andreas Brech and Espen Stang.

Work Requirements:

Attendance at the course, including lectures, demonstrations and laboratory exercises

Examination and assessment:

Home examination, graded pass or fail. The examination can be answered in Norwegian (or in another Scandinavian language) or English.

Supplementary exam:

There will be the option of one supplementary examination if a fail grade is achieved at the first attempt

Chosen book chapters as well as scientific articles, in total 320 pages.

Book (130 p.):

Gareth Griffiths. Fine structure immunocytochemistry. Springer-Verlag 1993. Chapters: 3, 7, 8, 9

Scientific articles (190 p.):

Albrecht, U., Seulberger, H., Schwarz, H. & Risau, W. Correlation of blood-brain barrier function and HT7 protein distribution in chick brain circumventricular organs. Brain research 535, 49-61 (1990).

Biel, S. S., Kawaschinski, K., Wittern, K. P., Hintze, U. & Wepf, R. From tissue to cellular ultrastructure: closing the gap between micro- and nanostructural imaging. Journal of microscopy 212, 91-99 (2003).

Bishop, D. et al. Near-infrared branding efficiently correlates light and electron microscopy. Nature methods 8, 568-570, doi:10.1038/nmeth.1622 (2011).

Colombelli, J. et al. A correlative light and electron microscopy method based on laser micropatterning and etching. Methods Mol Biol 457, 203-213 (2008).

Grabenbauer, M. et al. Correlative microscopy and electron tomography of GFP through photooxidation. Nature methods 2, 857-862, doi:10.1038/nmeth806 (2005).

Gruska, M., Medalia, O., Baumeister, W. & Leis, A. Electron tomography of vitreous sections from cultured mammalian cells. Journal of structural biology 161, 384-392, doi:10.1016/j.jsb.2007.10.008 (2008).

Knott, G. W., Holtmaat, A., Trachtenberg, J. T., Svoboda, K. & Welker, E. A protocol for preparing GFP-labeled neurons previously imaged in vivo and in slice preparations for light and electron microscopic analysis. Nature protocols 4, 1145-1156, doi:10.1038/nprot.2009.114 (2009).

Kukulski, W. et al. Correlated fluorescence and 3D electron microscopy with high sensitivity and spatial precision. The Journal of cell biology 192, 111-119, doi:10.1083/jcb.201009037 (2011).

Lucic, V. et al. Multiscale imaging of neurons grown in culture: from light microscopy to cryo-electron tomography. Journal of structural biology 160, 146-156, doi:10.1016/j.jsb.2007.08.014 (2007).

Micheva, K. D. & Smith, S. J. Array tomography: a new tool for imaging the molecular architecture and ultrastructure of neural circuits. Neuron 55, 25-36, doi:10.1016/j.neuron.2007.06.014 (2007).

Muller-Reichert, T., Srayko, M., Hyman, A., O'Toole, E. T. & McDonald, K. Correlative light and electron microscopy of early Caenorhabditis elegans embryos in mitosis. Methods in cell biology 79, 101-119, doi:10.1016/S0091-679X(06)79004-5 (2007).

Polishchuk, R. S. et al. Correlative light-electron microscopy reveals the tubular-saccular ultrastructure of carriers operating between Golgi apparatus and plasma membrane. The Journal of cell biology 148, 45-58 (2000).

Powell, R. D., Halsey, C. M. & Hainfeld, J. F. Combined fluorescent and gold immunoprobes: reagents and methods for correlative light and electron microscopy. Microscopy research and technique 42, 2-12, doi:10.1002/(SICI)1097-0029(19980701)42:13.0.CO;2-Y (1998).

Sartori, A. et al. Correlative microscopy: bridging the gap between fluorescence light microscopy and cryo-electron tomography. Journal of structural biology 160, 135-145, doi:10.1016/j.jsb.2007.07.011 (2007).

Schwarz, H. & Humbel, B. M. Correlative light and electron microscopy using immunolabeled resin sections. Methods Mol Biol 369, 229-256 (2007).

van Rijnsoever, C., Oorschot, V. & Klumperman, J. Correlative light-electron microscopy (CLEM) combining live-cell imaging and immunolabeling of ultrathin cryosections. Nature methods 5, 973-980, doi:10.1038/nmeth.1263 (2008).

Verkade, P. Moving EM: the Rapid Transfer System as a new tool for correlative light and electron microscopy and high throughput for high-pressure freezing. Journal of microscopy 230, 317-328, doi:10.1111/j.1365-2818.2008.01989.x (2008).

Watanabe, S. et al. Protein localization in electron micrographs using fluorescence nanoscopy. Nature methods 8, 80-84, doi:10.1038/nmeth.1537 (2011).