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Microscopy School Lesson 5 - Principles of Fluorescence Light Microscopy

Using transmitted light microscopy researchers can have an overview of the morphology of the specimen. However, the localization of specific structures and proteins inside the cell is not possible. Fluorescence microscopy came to fill this gap introducing the use of chemical dyes that allow the localization of intracellular structures. 

These dyes can be coupled to antibodies to recognize specific proteins, or have affinity for specific cellular structures such as DNA, mitochondria, etc. The discovery of fluorescent proteins pushed even further the development of fluorescence microscopy techniques and applications. Fluorescent proteins opened the door to live-cell fluorescence microscopy and its derivatives, such as FRAP, FRET, Photoactivation/Optogenetics, etc. Fluorescent proteins are also commonly used in other technologies that require more complex hardware such as TIRF.  

To be able to understand the full potential of fluorescence microscopy techniques and possible applications, it is crucial to have background knowledge of what is fluorescence and fluorescence microscopy. In this webinar, Dr Florindo will present the concept of fluorescence, and how is this physical propriety is applied to microscopy. We will give a brief overview of the hardware required for a fluorescent microscope, as well as discuss some concerns that should be considered when starting a fluorescence imaging protocol.

Overall, the goal is that individuals that attend this webinar gain (refresh) their knowledge about fluorescence microscopy potential. We hope to arm researchers with better tools to plan their work.

Learning Objectives:

  • Understand the principles of fluorescence.
  • Interpret the spectra of a given fluorophore.
  • Acquire an overview of the hardware  required for fluorescence microscopy.

Questions Answered:

  • How to choose a fluorochromes to avoid spectrum overlap?
  • Which filter should be chosen to image a given fluorophore?
  • What is photobleaching?

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