Section 2: Imaging Flashcards
Brightfield Microscopy
Outline the features of brightfield microscopy/a light microscope
- Light source
- Condenser lens to focus light on specimen
- Objective lens to collect light after it has passed through specimen
- Ocular/eyepiece lens to focus image onto eye
- Magnification is 40 to 1000x
- Only structures with a high refractive index (ability to bend light) are observable
Brightfield Microscopy
What is the meaning of resolution?
State the equation for resolution and what it means
Resolution: the ability to distinguish between two very closely positioned objects as separate entities
D = 0.61λ / nsinα
- D = resolution (smaller is better)
- λ = wavelength of the light (shorter is better)
- nsinα = numerical aperture; a property of the objective lens (higher is better)
- n = refractive index of medium between the specimen and objective lens (higher is better; use oil)
- α = the 1/2 angle of light entering the objective lens
Brightfield Microscopy
What does it mean to say ‘the resolution of a light microscope is 200 nm’?
A light microscope cannot resolve objects that are less than 200 nm apart
Describe phase contrast microscopy
- Used on live, unstained cells
- Certain parts of the cell (ex., nucleus) refract more light than others
- They slow the passage of a light beam by a quarter wavelength (1/4λ) due to their high refractive properties
- This causes interference (dimming of the light), making the nucleus and other refractive areas look darker (pictured)
- Phase contrast microscopy adds a phase plate behind the objective lens to shift another 1/4λ to increase contrast (total shift of 1/2λ)
Describe Normarski microscopy/differential intereference constrast microscopy
- Used on live, unstained cells
- Uses polarised light; microscope equipped with a polariser
- Interference of the polarised light generates the contrast
- Creates a 3D image
Fluorescence Microscopy
Describe fluorescence microscopy
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Used on fixed, permeabilised dead cells
- Necessary for fluorescent dyes/protein (fluorochromes) entry
- Fluorochromes are excited at their maximum absorbance for visualising
Fluorescence Microscopy
State 3 common fluorochromes
- DAPI or Hoechst to stain nuclei/DNA blue
- Microtracker Red to stain mitochondria
- Rhodamine-labeled phalloidin (molecule) to stain actin filaments red
Other dyes must be conjugated with antibodies to localise any molecules of interest in cells (immunofluorescent staining)
Fluorescence Microscopy
Describe the significance of GFP
- GFP/green fluorescent protein is derived from jellyfish
- GFP can be attached directly to the gene of interest in the expression plasmid
- It is transcribed and translated into a GFP-fusion protein
- The attachment site is at the end of the gene, and must be chosen carefully so as not to disrupt normal protein function
- GFP contains a short sequence of amino acids that are capable of fluorescing when excited with blue light (chromophore)
- By mutating various amino acids in GFP, new types of fluorescent proteins are created allowing two or more different fluorescent fusion proteins to be visualised
Allows for fluorescent imaging in live cells
Immunofluorescent Microscopy
Describe immunofluorescent microscopy
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Used on fixed, permeabilised dead cells
- Necessary for antibody entry
- Antibodies enter the cell and bind to specific proteins they are designed to recognise
- Secondary antibodies (“anti-mouse”) with attached fluorochromes are added and bind the primary antibody (“mouse monoclonal”)
- The secondary antibodies must be raised in a different animal than the animal which the monoclonal antibodies were obtained from
- Secondary antibodies (“anti-mouse”) with attached fluorochromes are added and bind the primary antibody (“mouse monoclonal”)
Immunofluorescent Microscopy
What are monoclonal antibodies?
- Special antibody hybrids
- They are a combination of myeloma cells and spleen cells; they are immortal cell lines that produce antibodies
- They are grown in HAT medium that selects for the hybrid cells
Describe dual label fluorescent microscopy
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Used on fixed, permeabilised dead cells
- Necessary for antibody entry
- Multiple images are taken at once, fluorescing each fluorochrome individually
- The images are digitally overlaid, allowing the viewing of multiple components of the cell at once
Describe laser scanning confocal microscopy
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Used on fixed, permeabilised dead cells
- Necessary for dye entry
- A moving laser light “scans” a specimen using mirrors and the specimen is reconstructed digitally
- The light is focused so that only a small portion of the specimen is illuminated at one time
- This prevents the whole cell from fluorescing at once
- Detecting fluorescence only from one focal plane produces a sharper image
Describe deconvolution microscopy
- Images are taken at different focal planes (Z-stacks) by means of a precisely controlled robotic microscope stage
- A math procedure removes fluorescence contributed from out-of-focus parts of the stained sample
- Uses the point spread function: deduces the underlying structure by comparison to a reference set of tiny fluorescent beads
- Final image is very detailed without blur
Describe two-photon excitation microscopy
Certain fluorochromes can be excited by a single photon (ex., 488 nm) or by two photons at half energy/twice the length (ex., 960 nm) to generate the same emission spectrum
- This trick is sued to explore thicker samples like deep tissue and focus the light at only one point to avoid scattered fluorescence
- No need for a pinhole to exclude out-of-focus light!
Describe FRET microscopy (fluorescence resonance energy transfer)
- Used to measure protein interactions in live cells
- CFP (cyan fluorescent protein) is excited
- If no protein interaction occurs then only cyan will fluoresce
- If protein interaction occurs then yellow will fluoresce
Electron Microscopy
Describe electron microscopy
- A wire filament serves as the electron source
- It is heated and the electrons move toward the anode
- A magnetic condenser (not glass like the others) focuses electrons on the specimen
- Specimen is stained with electron-dense heavy metals (lead, uranium, osmium tetroxide)
- Images are formed from electrons that:
- Pass through the sample (TEM - transmission electron microscopy)
- These samples must be chemically fixed, dehydrated, and embedded in plastic to allow for thin sectioning
- Or are scattered at the surface of the sample (SEM - scanning electron microscopy)
- Pass through the sample (TEM - transmission electron microscopy)
Electron Microscopy
Describe immunoEM
Antibodies added to samples can be detected using gold particles conjugated to Protein A
Protein A binds to Abs, and gold provides an electron dense image
