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Processing and Analysis of Fluorescence Microscopy Images

The course combines theory and practice on image processing focusing on fluorescence microscopy images. The course is divided in two parts, the first one presents the theory and methods to understand the algorithms for image processing included in most of the commonly used software packages such as Icy or Fiji. This part is taught by top researchers and developers working in the biomedical image processing field, using material and biological examples. The second part is a hands-on course where the student will applied the analyzed methods (or new ones) to their own image related research projects. This practical part will include the development of simple or more elaborated scripts or plugins, using the methods provided by the software packages.
We plan to populate the course with students from two different disciplines such as those with biological background and those trained in quantitative sciences such as mathematics, physics and informatics. The goal is to create small groups of students from different areas and combine their expertise to tackle a single problem.


February 29th to March 11th, 2016


Institut Pasteur de Montevideo

Mataojo 2020, Montevideo, 11400, Uruguay

Aim and objectives

The global aim of this course is to equip students to address fluorescence microscopy imaging questions from a comprehensive and quantitative perspective.
At the end of the course, the students should have gained basic abilities to:

  1. Precisely define a fluorescence microscopy imaging question and the type of data needed to answer it.
  2. Select an effective set of processing and analytical tools and apply them in a systematized and comprehensive manner.
  3. Design, modify or adapt a tool or set of tools to improve image processing and/or analysis performance.

The course is designed to foster two types of students: those with biological background and those trained in quantitative sciences such as mathematics and physics. Theoretical and practical sessions will be organized in a way that the skills of one group of students will help the other group.

Confirmed speakers and instructors

  • Alexandre Dufour, Institut Pasteur Paris, France
  • Stéphane Dallongeville, Institut Pasteur Paris, France
  • Steffen Hartel, Universidad de Chile, Chile
  • Victor Castañeda, Universidad de Chile, Chile
  • Mauricio Cerda, Universidad de Chile, Chile


Download the program in pdf file from here.

Invited talks

There will be two invited talks by Ulrich Kubitscheck with the following details.

Light Sheet Fluorescence Microscopy: A Pathway into New Worlds
Thursday, March 10, 11:00

Light sheet fluorescence microscopy is a relatively young light microscopic technique, which features optical sectioning, low photo-toxicity and rapid image acquisition. The technique is based on illuminating the sample orthogonally to the detection pathway with a thin, focused sheet of light. The basic principle of the method will be introduced, and current lines of methodological developments in our lab will be outlined: confocal light sheet imaging, 2-photon excitation, large volume imaging and combination with expansion microscopy.

Real Time Observation of Single Molecule Pathways in Biological Systems
Friday, March 11, 11:00

Ulrich Kubitscheck, Tim Kaminski, Katharina Scherer, Jan-Hendrik Spille, and Jan Peter Siebrasse*

Observation and tracking of fluorescently labeled molecules and particles in living cells reveals detailed information about intracellular processes on the molecular level, e.g. the nuclear export of RNA particles (1). Whereas light microscopic particle observation is usually limited to two-dimensional projections of short trajectory segments, we report here image-based real-time three-dimensional single particle tracking in an active feedback loop with single molecule sensitivity. We tracked particles carrying only 1–3 fluorophores deep inside living tissue with high spatio-temporal resolution (2). Using this approach, we succeeded to acquire trajectories containing several hundred localizations. We present statistical methods to find significant deviations from random Brownian motion in such trajectories. The analysis allowed us to directly observe transitions in the mobility of ribosomal (r)RNA and Balbiani ring (BR) messenger (m)RNA particles in living Chironomus tentans salivary gland cell nuclei. We found that BR mRNA particles displayed phases of reduced mobility, while rRNA particles showed distinct binding events in and near nucleoli (3).

(1) Siebrasse. J.P., T.Kaminski and U.Kubitscheck. 2012. Nuclear export of single native mRNA molecules
observed by light sheet fluorescence microscopy.  Proc Natl Acad Sci USA 109: 9426-31.

(2) Spille, J.-H., T.P. Kaminski, K. Scherer, J.S. Rinne, A. Heckel, and U. Kubitscheck. 2014 Direct observation of mobility state transitions in RNA trajectories by sensitive single molecule feedback tracking. Nucleic Acids Res. 2014; doi: 10.1093/nar/gku1194.

(3) Spille, J.-H. and U. Kubitscheck. 2015. Labelling and imaging of single endogenous messenger RNA particles in vivo. J. Cell Sci 128(20):3695-706.

More microscopy courses and workshops

After our course there will be held in Santiago, Chile, on March 14-21th 2016, the international course Optics, Forces and Development”. It will coincide with two symposia: “New frontiers in light sheet microscopy” (March 18th) and “In vivo approaches on brain research” (March 21st).

For more information and registration visit this webpage.