On completion of this module, students will have a comprehensive knowledge of the modalities of fluorescence microscopy available to life scientists. They will have understanding of the issues that need to be considered when designing light microscopy experiments, and will have insight into how to process samples for biological imaging experiments.

Lecture 1 - Overview of biological imaging
Introduction to module. Principles of biological imaging, basic history of imaging and instrumentation, temporal resolution, Nyquist, Abbe, basics of optics. Understanding of what makes an image, importance of bit depth, scale bars, use of colour.

Lecture 2 - Introduction to fluorescence and fluorescent markers
Principles of fluorescence, Jablonski diagrams, range of fluorescent dyes and chemicals, introduction to immunofluorescence using cultured cells, GFP and variants, DsRed and variants, basics of fluorescence light microscopy.

Lecture 3 - Use of fluorescent probes in cells
Modulation of gene and protein function in mammalian cells. Basics of cloning and expression of fluorescent constructs, DNA plasmids and their features. Strategies for gene downregulation in mammalian cells, RNA interference, shRNAs and siRNAs, experimental design. Introduction of fluorescent probes into mammalian cells, cell transfection techniques, microinjection of cells, electroporation, viral vectors. Fixation methods, use of antibodies and stains, immunofluorescence, blocking agents, mounting media.

Lecture 4 - Microscopy components
Microscope body configurations, objectives, optical elements, light sources, lasers, excitation and emission filters, dichroic mirrors, AOTFs, scanners, detectors, cameras, PMTs.

Lecture 5 - Confocal microscopy part I
Principles of confocal microscopy, wide-field versus confocal microscopy, optical sectioning, point scanner and Nipkow disk-based confocality. Fundamentals of confocal imaging, system set-up, pixel dwell time, image size, excitation and detection settings, Kalman averaging.

Lecture 6 - Confocal microscopy part II
Considerations for confocal microscopy of living cells. Effects of confocal setup on cell health and viability. Climate and environmental control during live cell imaging. Fundamentals of cell culture and types of cells available. Appropriate treatment of cells in live cell imaging experiments, use of media, buffers, climate control systems, issues relating to photo-toxicity and bleaching.

Lecture 7 - Confocal microscopy techniques part I
Introduction to cell and protein mobility techniques, live cell imaging, photobleaching, FRAP and FLIP. Introduction to protein-protein techniques in microscopy, FRET, FLIM, FCS, and FCCS.

Lecture 8 - Confocal microscopy techniques part II
Continuation of material from previous lecture.

Lecture 9 - Other light microscopy technologies
How to improve resolution in fluorescence microscopy, super-resolution, illumination based approaches (structured illumination (SI)), acquisition based approaches (STED, PALM, STORM). Principles of light sheet (LS) microscopy, principles of multi-photon microscopy, principles of TIRF microscopy.

Lecture 10 - High content screening (HCS) and analysis (HCA)
Principles of automated microscopy, technologies involved, instrumentation available, limitations, autofocus, image acquisition, cell-based screening, basic principles of image analysis, image and data storage.

Lecture 11 - Image analysis
Overview of image processing, basics of image analysis and quantification, object segmentation, tools and software available, image presentation.

Lecture 12 - Analysis of co-localisation in cells
Appropriate image acquisition parameters for co-localisation experiments, importance of pixel intensity distribution and saturation. Principles of co-localisation, co-occurrence, correlation, existing methods including Pearson correlation, Manders coefficient, Costes mask, Rank-weighted co-localisation, object-based co-localisation methods.

Students taking this module should have a basic understanding of cell form and function, particularly with respect to the organisation of subcellular structures.

• Feedback individually to students, post-assessment

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