L9: Microscopy Flashcards
What are the parts of a microscope?
→detector →objective →specimen →light conditioning system →light source
What can the light source be?
→halogen
→XBO
Describe the light microscopic specimen
→cover glass
→sample surrounded by embedding medium
→glass slide
What is the life imaging part of a microscope?
→ the box prevents focus instability
How is CO2 atmosphere maintained?
→controller allows adjustment of air flow and %CO2
→air tight table top encloses live cell culture
Which cell processes have the shortest experimental time scale?
→microtubules
→cytoskeleton
Which cell process have the longest experimental timescales?
→differentiation
→development
What is the triangle of frustration?
→user have to find the balance between temporal resolution, spatial resolution, and sensitivity
What is temporal resolution?
→low exposure time, low pixel number, binning
What is spatial resolution?
→high pixel number, no binning
What is sensitivity?
→ high exposure time, binning
What are the markings on objectives?
→magnification →application →coverslip thickness →working distance →immersion medium/numerical aperture
What does the aperture of objective determine?
→the resolution
What does a high aperture mean?
→The higher the numerical aperture the better the resolution power of the objective.
What are the fundamentals of the light microscope?
→Brightfield, DIC, Phase
What are the three stages of light microscopy?
1) Histology-
2) Phase contrast
3) Time-lapse
How can you locate the protein of interest in histology?
→Using Antibodies
What is the role of histology in light microscopy?
→tissue organisation
What is the role of phase contrast in LM?
cell morphology
What is a problem with using phase contrast in LM?
→Can’t determine how changes in morphology went about
What is the role of time-lapse in LM?
→heart cell differentiation, cell migration
What are the two types of EM?
Transmission EM
Scanning EM
What is the difference between TEM and SEM?
→TEM examine the internal structures of a cell, whereas ascanning electron microscope only allows visualization of the surface of a structure.
Why can we use electrons to visualise structures?
Electrons find it difficult to go through high material areas=darker
What are the two types of LM set-up?
→bright filed
→fluorescence
What are the differences between bright field and fluorescence microscopy?
→Fluorescence= source of light(laser beam) goes through filter cube and emits light source to sample,
Why do emitted light shift to longer wavelength?
→due to loss of energy
What is photobleaching?
→process whereby fluorophores lose their ability to fluoresce effectively due to high intensity illumination
How do you reduce photobleaching?
→use shorter exposure
→reduced excitation light intensities
What are fluorescent proteins?
→GFP
→ naturally found in light producing cells of cnidarians
How are live studies of fluorescent tags carried out in cells?
→fluorescent proteins can be fused with other proteins and introduced in cells via transfection
What is the difference between antibodies and protein fusion for fluorescence?
→Antibodies method- cells have to be fixed- when not alive
What are the two types of visualising fluoescence?
→antibodies
→protein fusion, tag the gene
What are the two types of focuses in microscopy?
→confocal
→widefield
What is a difference between confocal and widefield microscopy?
→widefield microscope, the entire focal volume is illuminated, but that creates blur from areas out of focus above and below the image plane
→confocal microscopescans a sample with a focused beam of light, higher resolution
Compare confocal and widefield microscopy
→confocal has a higher z-resolution and reduced out of focus blur- clearer and crisper
→ only a small volume can be visualised with confocal
What do bigger volumes need in order to be viewed by confocal microscope?
→need time consuming sampling and image reassembling
What can be viewed with intracellular live imaging?
→Microtubule dynamics
(GFP-tubulin)
→Vesicle transport through microtubules
(GFP-Kinesin)
What are the two types of 3D reconstruction?
→non-neoplastic
→neoplastic
