Microscopy School Lesson 8 - Lifting the Cellular Fog with Total Internal Reflection Fluorescence (TIRF) Microscopy

Total internal reflection fluorescence (TIRF) microscopy is a decades-old, camera-based imaging technique which excels at viewing and highlighting the basal surface or interface of cellular specimens with exquisite axial resolution, on the order of 5-6 times greater than the axial resolution offered by confocal microscopy. The optics required to achieve this axial resolution are fairly straightforward, and as such the technique has been instrumental in elucidating key features about fundamental biological processes such as vesicle transport and fusion/excretion, lipid, protein, and cytoskeletal membrane interactions, cell adhesion, and viral capsid assembly dynamics to name a few. In more recent years, TIRF has been instrumental for advanced techniques such as single-molecule imaging and super-resolution microscopy.

In this microscopy course lecture Dr John Oreopoulos covers the basics of TIRF microscopy, the necessities that make for a successful TIRF imaging experiment, the challenges of TIRF image analysis, and discusses how TIRF microscopy in combination with other imaging modalities – either simultaneously or sequentially – have advanced our understanding of complex cellular processes and molecular dynamics. Some of the latest developments of this technique will also be reviewed.

Key Learning Objectives:

• Understand the concept of total internal reflection and how it is implemented in the fluorescence microscope.
• Comprehension of the technical requirements for a successful TIRF imaging experiment.
• Gain awareness of the strengths and limitations of TIRF microscopy as compared to confocal microscopy.
• Recognition of the types of applications that benefit the most from TIRF imaging.