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BIOL Exam I - Ex. 3
Advanced Techniques of Light Microscopy
| Question | Answer |
|---|---|
| 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 |
| 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 |
| 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 |
| 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 |
| Waves emitted are always ___ than waves absorbed. | longer |
| 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 |
| What fluorophore directly stains cellular components? | - DAPI - it directly binds to A-T rich regions in DNA - direct fluorescence |
| What fluorophore conjugates to non-fluorescent molecules that bind to cellular components? | - Texas Red - it attaches to phalloidin, which binds to actin proteins |
| autofluorescence | occurs due to endogenous (growing/originating from within an organism) biomolecules that fluoresce, such as chlorophyll, lignin, and carotene |
| Chlorophyll has an absorption band in the ___ and ___ excitation regions (around 450 nm) and emits ___ light (around 680 nm). | - blue and green - red |
| What is immunofluorescence? What's an example of this? | - - BODIPY FL |
| 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 |
| 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 |
| 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 |
| The fluorescence microscopes we will use expose slide with light of specific wave lengths (___, ___, ___) from above. | - UV - Blue - Green |
| 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 |
| What color of light do we want a DAPI excitation filter to pass? | UV only |
| photobleaching | |
| 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 |
| What are diatoms? Describe them. | - microscopic algae - enclosed in a shell made of silica - ornate; very pretty looking |
| 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 |
| 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 |
| fluorophores | fluorescent molecules used to stain specimens |
| 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) |
| 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 |
| The component of an optical microscope that is altered most in converting from brightfield optics to darkfield or phase contrast optics is the ___. | condenser |
| 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 |
| 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 |
| Which type of light would be required to excite a fluorochrome that fluoresced blue? | UV |
| The use of antibodies in fluorescence microscopy is a technique known as ___. | immunofluorescence |
| In exercise 3, buccal cells stained with ___ were observed using brightfield microscopy. What cell structures were made visible? | - methylene blue - nucleus, cytoplasm, plasma membrane |
| In exercise 3, cheek cells stained with ___ and ___ were viewed under fluorescent microscopy. | - DAPI - Texas Red®-Phalloidin |
| 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 |
| What are advantages and disadvantages of brightfield microscopy (with staining)? | - stain provides better contrast - BUT specimens are killed - artifacts from the stain are added |
| 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 |
| 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 |
| 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 |
| 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 |
| 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 |
| 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 |
| 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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jessica.gvc
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