Visualizing cells and their Molecules Flashcards
What is the size range of:
A. cell
B. organelles
C. molecules
A. 1-100 micrometers
B. 1-2 micrometers
C. 2-20 nanometers
What are the main components of a fluorescent microscope? Explain.
- Light Source: Provides the excitation light.
- Excitation Filter: Selects the wavelength of light that excites the fluorescent molecules.
- Beam Splitter: Directs the excitation light towards the sample and allows emitted light to pass through to the detector.
- Objective Lens: Focuses the excitation light onto the specimen and collects the emitted fluorescence.
- Emission Filter: Allows only the emitted fluorescence light to reach the detector while blocking other wavelengths.
- Detector: Usually, a camera or the human eye records the light emitted from the sample.
What is the purpose of CLSM, and when is it preferable over regular fluorescence microscopy?
Confocal Laser Scanning Microscopy
eliminates out-of-focus light, making it ideal for thick samples and 3D reconstructions. It’s preferable when clear, detailed images of thick specimens are needed.
What are the criteria for choosing between live imaging and fixed samples?
Live imaging should be used for dynamic processes (e.g., cell division, protein trafficking), and fixed samples should be used for high-resolution snapshots or when live processes aren’t needed.
How can you use imaging to test if two proteins bind to each other?
Use FRET to detect proximity between proteins (1-10 nm) or BiFC (bifluorescence Complementation) to visualize interactions directly.
What are the advantages and disadvantages of FRET for protein interaction studies?
Advantages: Sensitive, allows live-cell imaging, quantitative data.
Disadvantages: It requires specific fluorophores and is sensitive to orientation and distance.
What imaging technique would you use to test if two compartments are connected?
Use FRAP (Fluorescence Recovery After Photobleaching) to observe protein diffusion and compartment continuity.
What are the applications of AFM, and how does it work?
AFM (Atomic Force Microscopy) is used for high-resolution surface imaging and force measurements. It works by scanning a sample with a sharp probe and detecting deflections caused by interactions with the surface.
What does “FRET” stand for, and how does it work?
FRET stands for Fluorescence Resonance Energy Transfer. It works by transferring energy from a donor fluorophore to an acceptor when they are within 1-10 nm, indicating molecular proximity.
What are the applications of TIRF, and how does it work?
TIRF (Total Internal Reflection Fluorescence) is used for single-molecule detection and events near the plasma membrane. It excites fluorophores in a thin section (~100-200 nm) by generating an evanescent field at the glass/sample interface.
What is one application of FRET other than protein-protein interaction?
FRET can be used as a Ca2+ sensor, where a conformational change upon calcium binding brings fluorophores close enough for energy transfer, generating a measurable signal.
