Advanced Techniques of Light Microscopy
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| What is "in vivo" and what is an example of this technique? | - "within the living"
- the structure or process of interest is studied within the living cell; whole, living organisms
- animal testing, clinical trials, observing live cells' nuclei with bright field
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| What is "in situ" and what is an example of this technique? | - "on site"
- the structure or process of interest is in its natural location, but perhaps not under normal conditions
- examining a cell within a whole organ intact and under perfusion; examining a cell within a removed leaf
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| What is "in vitro" and what is an example of this technique? | - "within the glass"
- the structure of interest or cell is separated from its normal biological location; partial or dead organisms
- cell cultures, test tubes; observing chloroplast removed from a plant cell
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| A fluorophore is a molecule which has the property of fluorescence. It absorbs electromagnetic radiation of some ___ and then emits radiation of some ___ wave length. | - specific wave length
- slightly longer, lower energy
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| Waves emitted are always ___ than waves absorbed. | longer
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| What are the two ways fluorophores are used to mark locations of components of interest? | - some stain (bind to) cellular components DIRECTLY
- others can be attached (CONJUGATED) to non-fluorescent molecules that bind to cellular components
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| What fluorophore directly stains cellular components? | - DAPI
- it directly binds to A-T rich regions in DNA
- direct fluorescence
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| What fluorophore conjugates to non-fluorescent molecules that bind to cellular components? | - Texas Red
- it attaches to phalloidin, which binds to actin proteins
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| autofluorescence | occurs due to endogenous (growing/originating from within an organism) biomolecules that fluoresce, such as chlorophyll, lignin, and carotene
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| Chlorophyll has an absorption band in the ___ and ___ excitation regions (around 450 nm) and emits ___ light (around 680 nm). | - blue and green
- red
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| What is immunofluorescence? What's an example of this? | -
- BODIPY FL
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| What's the difference between direct and Indirect immunofluorescence? | - in direct, the fluorophore binds directly to the primary antibody of a (tubulin) protein
- in indirect, the fluorophore binds to a secondary antibody, which is attached to the primary antibody of a protein; like the BODIPY we used
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| What are the advantages/disadvantages of direct immunofluorescence? | - shorter sample staining times
- simpler dual and triple labeling procedures
- BUT lower signal
- higher cost
- less flexibility
- more difficult labeling procedure
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| What are the advantages/disadvantages of indirect immunofluorescence? (We used this one) | - greater amplification of the signal
- inexpensive, available in wider variety
- BUT potential for cross-reactivity
- need to find primary antibodies that are not raised in the same species
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| The fluorescence microscopes we will use expose slide with light of specific wave lengths (___, ___, ___) from above. | - UV
- Blue
- Green
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| Light that excites fluorophores enters specimen via ___. Molecules in cells absorb the light and re-emit the light at a ___, ___ energy wave length. What is observed is just the ___. Everything else remains black. | - the objective
- longer, lower
- fluorescence
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| What color of light do we want a DAPI excitation filter to pass? | UV only
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| photobleaching |
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| What is the problem with stains? | - specimens prepared with stains usually are killed
- stains may add artifacts to cell structures that don't normally exist in the live cells
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| What are diatoms? Describe them. | - microscopic algae
- enclosed in a shell made of silica
- ornate; very pretty looking
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| How does darkfield microscopy work? What does the image end up looking like? | - light approaches the specimen at a very wide angle; only light that has been scattered by a specimen will reach the objective (if no specimen, no light, only see black)
- everything is black except for what the specimen has reflected, which is bright
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| How does phase contrast microscopy work? What does the image end up looking like? | - a transparent ring allows only a hollow cone of light to reach the specimen; light that did not interact w/ specimen is weakened by a phase plate in the objective
- you see some background light but also light that has been diffracted by the specimen
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| fluorophores | fluorescent molecules used to stain specimens
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| How does brightfield microscopy work? What does the image end up looking like? | - produces a bright background field of view
- specimens visible when they absorb, refract, or reflect light (thereby diminishing their brightness = contrast)
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| The "slider" of the Olympus CX31 student microscope condenser provides the ability to perform what types of microscopy? | - light microscopy
- brightfield microscopy
- darkfield microscopy
- phase contrast microscopy
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| The component of an optical microscope that is altered most in converting from brightfield optics to darkfield or phase contrast optics is the ___. | condenser
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| Toothpicks used for collection of human cheek cells are considered to be a(n) ___ and would be most safely discarded in the ___. | - biohazard
- biohazard waste beaker
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| The property of some compounds to absorb light at a given wavelength and shortly thereafter emit light at a slightly longer wavelength is called ___. | fluorescence
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| Which type of light would be required to excite a fluorochrome that fluoresced blue? | UV
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| The use of antibodies in fluorescence microscopy is a technique known as ___. | immunofluorescence
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| In exercise 3, buccal cells stained with ___ were observed using brightfield microscopy. What cell structures were made visible? | - methylene blue
- nucleus, cytoplasm, plasma membrane
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| In exercise 3, cheek cells stained with ___ and ___ were viewed under fluorescent microscopy. | - DAPI
- Texas Red®-Phalloidin
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| What are advantages and disadvantages of brightfield microscopy (without staining)? | - can observe living specimens
- simple and easy to set up
- BUT low contrast
- limited resolution
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| What are advantages and disadvantages of brightfield microscopy (with staining)? | - stain provides better contrast
- BUT specimens are killed
- artifacts from the stain are added
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| What are advantages and disadvantages of phase contrast microscopy? | - can observe living specimens
- high contrast, resolution
- ideal for viewing thin specimens
- BUT doesn't work well with thick specimens
- distortions/artifacts can occur
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| What are advantages and disadvantages of darkfield microscopy? | - can observe living specimens
- high contrast
- ideal for observing external details
- BUT can kill specimens if light too strong
- doesn't work well with thick specimens
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| What are advantages and disadvantages of fluorescent microscopy? | - intracellular structures can be highlighted using diff. wavelengths
- high contrast, resolution
- versatile
- BUT specimens are killed
- photobleaching occurs
- artifacts can be added by FIX and PERM solutions
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| In exercise 3 you prepared a wet mount of human cheek cells by adding solutions to your sample of cheek cells. What does the FIX solution contain? What is the purpose of it? | - paraformaldehyde in a buffered solution
- the purpose of this toxic solution is to kill the cells while also preserving their positions from when they were alive; does this by cross linking cellular structures
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| In exercise 3 you prepared a wet mount of human cheek cells by adding solutions to your sample of cheek cells. What does the PERM solution contain? What is the purpose of it? | - a mild buffered detergent
- this solution makes it easier for dye to enter cells by dissolving their cellular membranes
- if the PERM solution was forgotten, then the fluorophores would not be able to permeate the cells
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| In exercise 3 you prepared a wet mount of human cheek cells by adding solutions to your sample of cheek cells. What does the DAPI/Texas Red-Phalloidin solution contain? What is the purpose of it? | - the DAPI/Texas Red-Phalloidin solution contained toxic fluorescent stains
- their purpose is to shine when excited, allowing us to observe cellular structures
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| You observed BPAE cells using fluorescent microscopy. What stains were used to prepare these cells? | - Texas Red-Phalloidin; this dye labels the microfilaments of cytoskeletons
- Green fluorescence; microtubules are shown using green fluorescence
- Blue-fluorescent DAPI; using this dye allows you to see DNA in nuclei
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