BIOL40560 Biological Imaging

Academic Year 2021/2022

In the life and biomedical sciences the form and function of organisms, particularly at the cellular and subcellular levels, is often studied through imaging and microscopy approaches. This module informs about the concepts of imaging and microscopy; the importance of resolution and its limits; optical components; and microscope design. Students will gain understanding of widefield microscopy, laser scanning confocal microscopy, spinning disk confocal microscopy; super-resolution microscopy, automated microscopy; and techniques associated with each. This module will also discuss live cell imaging and photobleaching methods to determine molecule function. Cell culture and types of cells available, treatment of cells in live cell imaging experiments, use of media, buffers, climate control systems, and issues relating to photo-toxicity will also be explored. Consideration will also be given to fluorescent proteins, fixation methods, use of antibodies and stains, immunofluorescence, and mounting media. A basic introduction to the analysis of biological images will also be given. This module also provides practical exposure to concepts in imaging through lab-based workshops.

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Curricular information is subject to change

Learning Outcomes:

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.

Indicative Module Content:

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.

Student Effort Hours: 
Student Effort Type Hours




Autonomous Student Learning




Approaches to Teaching and Learning:
The module is delivered through a series of lectures and small group practical (lab-based) workshops that will provide students the opportunity to see various types of microscopy systems and imaging software in action. 
Requirements, Exclusions and Recommendations
Learning Recommendations:

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

Module Requisites and Incompatibles
CNWY40120 - Advanced Biological Imaging, CNWY40170 - Fundamental Biological Imaging

Assessment Strategy  
Description Timing Open Book Exam Component Scale Must Pass Component % of Final Grade
Assignment: The assessment will take the form of a series of short-answer problem-based learning questions. Varies over the Trimester n/a Graded No


Carry forward of passed components
Remediation Type Remediation Timing
In-Module Resit Prior to relevant Programme Exam Board
Please see Student Jargon Buster for more information about remediation types and timing. 
Feedback Strategy/Strategies

• Feedback individually to students, post-assessment

How will my Feedback be Delivered?

Not yet recorded.

Timetabling information is displayed only for guidance purposes, relates to the current Academic Year only and is subject to change.
Workshop Offering 1 Week(s) - 8 Fri 10:00 - 10:50
Workshop Offering 1 Week(s) - 7, 8 Fri 13:00 - 13:50
Workshop Offering 1 Week(s) - 7 Fri 15:00 - 15:50
Lecture Offering 1 Week(s) - 5, 6, 7, 8 Mon 14:00 - 14:50
Workshop Offering 1 Week(s) - 5, 6, 7 Thurs 11:00 - 11:50
Lecture Offering 1 Week(s) - 5, 6, 7 Tues 10:00 - 10:50
Workshop Offering 1 Week(s) - 8 Wed 12:00 - 12:50
Workshop Offering 1 Week(s) - 8 Wed 14:00 - 14:50
Lecture Offering 1 Week(s) - 6, 7 Wed 15:00 - 16:50