Imaging 2

Microscopy images contain a wealth of information. Extracting this information in a way that is not subject to error requires 1) applying of the physics of image formation to assess the limits of physical information present; 2) applying of the mathematical concepts underlying digital image formation and analysis to allow for objective and complete information extraction; 3) application of the concepts of different microscopy methods to assess what method should be used to obtain a specific type of information and 4) the ability to automate image analysis tasks by using a computer and appropriate methods to efficiently, accurately and reproducibly extract objective, complete and physically relevant information.

In this course the student will apply the knowledge obtained in imaging 1 in practice. The student will learn to analyse complex imaging data from relevant papers, and perform experiments involving advanced microscopy concepts.

The student will be introduced to advanced imaging and image analysis concepts, among others Single molecule localization Microscopy (SMLM), Total internal reflection (TIRF), Structured Illumination microscopy (SIM), fluorescence recovery after photobleaching (FRAP), tracking, registration, data visualization and neural networks.

These advanced concepts, as well as the basic concepts from imaging 1, will be brought into practice as the student will be guided through the most important, up-to-date fluorescence microscopy techniques for quantitatively studying cellular function and dysfunction at the nanoscopic scale. The students will work (in groups of five) on various topics (four will be chosen from a total of six) using different advanced fluorescence bioimaging techniques: cell division (mitosis and meiosis) will be studied using confocal time lapse microscopy and single molecule localisation (SMLM) (topics 1, 2), the assembly of microtubules will be studied using total internal reflection (TIRF) microscopy and the role of focal adhesions in migration using structured illumination microscopy (SIM) (topics 3, 4), gene transcription regulation will be studied by fluorescence recovery after photobleaching (FRAP) (topic 5), and cell membranes will be visualised and cell volume/surface ratios will be determined using specific membrane and nuclear staining and 3-D confocal microscopy (topic 6). For each topic the students will use the various state-of-the-art research microscopes to record images themselves (under supervision), will analyse the data, will write a report and in a final session will discuss the reports with the supervisor in a 15 minute session.

Topics not dealt with in the practical sessions will be dealt with in the review of relevant papers, where students will be asked to use the advanced concepts introduced to extract relevant info and discuss the paper and its methodologies.

The course is closely related to all Nanobiology courses as a variety of cell biological model systems are investigated using microscopes, analysed using mathematical techniques and reported in writing and discussed afterwards