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Questions and Answers
What is the tri-peptide sequence responsible for GFP fluorescence?
What is the effect of changing Serine 65 to Threonine in the GFP sequence?
What is the effect of changing Tyrosine 66 to Tryptophan in the GFP sequence?
Where is the GFP gene usually inserted to maximize the chance of correct processing of the fusion protein?
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What is the limitation of using fluorescent proteins due to high intensity laser light?
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What is the limitation of using fluorescent proteins due to the handling of excess protein by the cell?
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What is the limitation of using fluorescent proteins that affects the function of the fusion protein?
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What is the type of microscope that allows cells to be viewed with nearly as much detail as an electron microscope?
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Who were awarded the Nobel Prize in Chemistry in 2014 for the development of super-resolved fluorescence microscopy?
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What is the primary function of the collector lens in a modern microscope?
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What is the approximate resolution limit of a standard light microscope?
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What is the primary function of the condenser lens in a microscope?
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What type of microscopy uses electromagnetic lenses instead of optical lenses?
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What is the primary limitation of electron microscopy?
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What is the primary function of fluorophores in fluorescence microscopy?
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What is the primary difference between the original form of GFP and its mutants?
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What is the typical combination of stains used in histology?
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Why do electron microscopes have a higher resolution than light microscopes?
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What is the main limitation of standard light microscopes?
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What is the role of the collector lens in a modern microscope?
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What is unique about the original form of GFP?
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What is the primary advantage of electron microscopes over light microscopes?
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What is the purpose of fluorophores in fluorescence microscopy?
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What is the typical combination of stains used in histology?
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What is the primary limitation of fluorescence microscopy?
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What is the role of the condenser lens in a microscope?
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Why can't electron microscopes produce colour images?
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What is the primary cause of phototoxicity in fluorescent microscopy?
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What is the effect of inserting the GFP gene after the start codon?
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What is the primary advantage of super-resolution microscopes?
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What is the effect of changing Tyrosine 66 to Tryptophan in the GFP sequence?
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What is the primary reason for overexpression of fluorescent proteins?
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What is the primary limitation of using fluorescent proteins due to protein folding?
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What is the result of changing Serine 65 to Threonine in the GFP sequence?
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What is the primary characteristic of red fluorescent proteins?
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What is the primary application of super-resolution microscopes?
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Study Notes
GFP and Fluorescence
- GFP fluorescence arises from the tri-peptide sequence Ser65-Tyr66-Gly67.
- Substituting Serine 65 with Threonine significantly enhances fluorescence and photostability while shifting emission into the green spectrum.
- Replacing Tyrosine 66 with Tryptophan results in a shift towards blue fluorescence.
- GFP can label either the N-terminus or C-terminus of a protein, depending on insertion location relative to the coding sequence.
- C-terminus labeling is preferred for improved fusion protein processing.
- Red fluorescent proteins (RFP) have been discovered in coral, expanding the fluorescent protein spectrum from blue to dark red.
Limitations of Fluorescent Proteins
- Photobleaching leads to irreversible loss of fluorescence due to intense laser exposure.
- Phototoxicity can occur, causing cellular damage from high-intensity light.
- Overexpression issues arise as tagged genes may be expressed excessively, altering normal cellular handling of proteins.
- Protein folding can be impacted by fluorescent protein tags, potentially disrupting function.
Microscopy Advances
- Super-resolution microscopes provide detailed cellular imaging similar to electron microscopy capabilities.
- The 2014 Nobel Prize in Chemistry was awarded to Eric Betzig, Stefan W. Hell, and William E. Moerner for contributions to super-resolved fluorescence microscopy.
Microscopy Principles
- Microscopes utilize lenses to magnify light passing through samples.
- A built-in light source is focused through a collector lens and mirror onto a specimen via a condenser lens.
- Standard light microscopy has a resolution limit of approximately 200 nm, insufficient for visualizing smaller cellular structures and viruses.
Staining Techniques
- Hematoxylin and Eosin staining is the most common method in histology; hematoxylin stains acidic components like the nucleus, while eosin targets basic structures such as cytoplasm and cell walls.
Electron Microscopy
- Electron microscopes apply similar principles as light microscopes but utilize electrons for imaging, enabling visualization of smaller objects due to their shorter wavelength.
- They cannot render color images and are unsuitable for live cell observation due to vacuum requirements.
Evolution of GFP
- Original GFP forms were optimized for aquatic environments and existed as a tetramer with low fluorescence and high susceptibility to bleaching.
- Mutants of GFP have been engineered to fold correctly and function efficiently at mammalian body temperature (37 degrees Celsius).
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Description
Learn how microscopes work and their limitations. Understand how lenses and light sources are used to magnify and visualize biological samples. Discover the minimum size limit of standard light microscopes.
