Microscopy – Bio-Imaging, 7.5 cr
There is a dramatic development of microscopical methods for visualization of biological structures and physiological events. This development runs in parallel with the explosive progress in molecular biology and protein biology (genomics and proteomics). It is important to be able to study where, and under which conditions, identified genes are expressed and where proteins are localized in cells and organisms.
Figure 1. Immunofluorescence is a method commonly used for the visualization of cells in culture or in histological sections of tissues and organs. (A) In culture, cells from neonatal rat pineal organs differentiate into neuronal types with different characters: red cells contain neuron-specific cytoskeletal proteins, while green cells contain serotonin. (B) In a histological section of the photosensory pineal organ of a salmon, photoreceptor cells show differential localization of serotonin (blue), the phototransduction protein arrestin (red), and protein component of the photopigment (opsin; green).
The course focuses on microscopy-based methods used in today's research, with special emphasis on methods in which fluorescent markers are used. There is an enormous variety of fluorescent markers that can be used to label specific biomolecules, to label cells that express specific genes or to visualize physiological events in cells. For example, it is possible to label the neurons in the brain that express the gene for the rate-limiting enzyme in dopamine biosynthesis, the neurons that contain dopamine, the neurons which contain cell membrane receptors binding to dopamine, the neurons responding to dopamine, and the mechanism by which these neurons respond - all with different fluorescent markers! For two more examples, see figure 1 andfigure 2.
Figure 2. These are hyphae formed by a bacterium called Streptomyces coelicolor. A protein located at the hyphal tips was detected with immunofluorescence and a specific antiserum (green). It is necessary to partially degrade the cell walls with lysozyme in order to get the antibodies into the cells. The peptidoglycan cell walls were labeled using the lectin wheat germ agglutinin conjugated to a fluorophore (blue). The DNA was detected by staining with 7-aminoactinomycin D (red).
The course gives a theoretical background to the most commonly used microscopic methods, the different types of biomarkers, and the optimization of microscopic samples. It includes a practical project where the students prepare and analyze microscopic samples with fluorescent markers.
The course also contains demonstrations of advanced research microscopes. It deals with the basic theory of methods that improve resolution - especially depth (z) resolution - and the visibility of small structures at the limit of optical resolution. Here, emphasis is laid on deconvolution microscopy (figure 3) and confocal laser scanning microscopy (figure 4). The course also includes a demonstration of a "live-imaging" microscopy system, during which physiological events are visualized in living cells.
The course is suitable for those who consider a career in biomedical sciences in an academic environment or in the industry.
Figure 3. Fluorescence microscopy images are usually blurred by diffracted light or light from parts of the preparation that are not in focus. The image to the left shows an example of this. It is an Arabidopsis cell in which the microtubules have been visualized by immunofluorescence microscopy and an antibody against beta-tubulin. Using a technique called image deconvolution, right, it is possible to approximately resassign diffracted and out-of-focus light to its point of origin and thereby generate sharp images. Images kindly provided by Mari Aidemark, Department of Cell and Organism Biology.
Figure 4. If you view a thick (0.1 mm) histological section in a wide-field fluorescence microscope (WF; left) it is difficult to discern structures even in a strongly fluorescent specimen. This is because you see light from all planes of the section, but the focal plane (where you see structures sharply) is thin. With a confocal microscope (CLSM; right) only light from the focal plane is registered and you see a thin "optical section" with distinct structures, in this example photoreceptor cells in the retina of a cichlid fish.
Another figure. An old-fashioned (but still scientifically useful) variant of bio-imaging: a neuron that has been stained with a so-called Golgi impregnation. With this method, single neurons are stained in their entirety. Camillo Golgi, who discovered the method, received the Nobel Price in medicine 1906, together with Santiago Ramón y Cajal, for studies of the structure of the nervous system!
Education office, Ecology building
Telephone: +46 46-222 73 16
E-mail: Christina.Ledje [at] biol.lu.se
Molecular Cell Biology
Telephone: +46 46-222 85 84
E-mail: Klas.Flardh [at] biol.lu.se
Spring term 2017:
Guy Cox (2012) Optical Imaging Techniques in Cell Biology, 2nd edition, CRC Press., ISBN: 978-1-4398-4828-9. Available as eBook for registered students.
The course is offered during the second part of the spring term.
The language of instruction is English.