In this iBiology lecture, Dr. Nico Stuurman (UCSF, HHMI) provides an informative introduction to the topic of Fluorescence Microscopy. Dr. Stuurman begins with the motivation for using Fluorescence in microscopy: the ability to perform high contrast, quantitative live cell imaging with a high degree of specificity. He then shows, via simple experiments that one can do at home – or your local pub – what fluorescence is. After providing essential definitions and simple explanations, he describes the concepts of excitation and emission spectrum. He then ties it all together by explaining the energy absorption and emission in the form of Jablonski diagrams.
In the second half of the video, Dr. Stuurman shows (schematically) how a Fluorescence Microscope works, and delves into the topic of interference filters that are a key component in modern Fluorescence Microscopes. He then shows where these components are present in a real-world microscope in his lab. After a brief visit to the lab, Dr. Stuurman also discusses the phenomenon of photobleaching and how one can mitigate the impact of photobleaching in Fluorescence Microscope experiments.
In this iBiology video, Nobel Laureate Dr. Roger Tsien (RIP) provides an excellent background on the topic of Green Fluorescent Protein (GFP).
Some organisms produce what has been named Green Fluorescent Protein (GFP), which emits a shimmering light. The formation of GFP is regulated by a gene that can be incorporated into the genomes of other organisms. Because GFP can be linked to other proteins thanks to genetic engineering, it has become an important tool for studying biological processes in cells. During the 1990s, Roger Y. Tsien elucidated how GFP produces its shimmering light and succeeded in varying the color of the light so that different proteins and multiple, simultaneous biological processes could be tracked.
In this video, Dr. Jennifer Waters (Director of the Nikon Imaging Center at Harvard Medical School) describes how fluorescence microscopy works, using explanatory animations. She details the method of selecting filters that will allow the optimal collection of light from fluorophores, while reducing background and increasing contrast. This video is part I of a two-part video series.
In this video titled Dr. Jennifer Waters (Director of the Nikon Imaging Center at Harvard Medical School) elaborates on “Imaging multiple fluorophores and dealing with bleed-through”. This video is part II of a series and it is best to view part I before viewing part II. In the video, Dr. Waters walks you through best practices for imaging more than one fluorophore, including critical controls experiments, why bleed-through can occur when imaging multiple fluorophores and various strategies for reducing bleed-through.
Cameras selection for Fluorescence Microscopy (very low light): sCMOS Cameras
Camera selection for Fluorescence Microscopy (low light): CMOS Cameras with low read-noise