Busy. Please wait.
or

show password
Forgot Password?

Don't have an account?  Sign up 
or

Username is available taken
show password

why


Make sure to remember your password. If you forget it there is no way for StudyStack to send you a reset link. You would need to create a new account.
We do not share your email address with others. It is only used to allow you to reset your password. For details read our Privacy Policy and Terms of Service.


Already a StudyStack user? Log In

Reset Password
Enter the associated with your account, and we'll email you a link to reset your password.

Remove Ads
Don't know
Know
remaining cards
Save
0:01
To flip the current card, click it or press the Spacebar key.  To move the current card to one of the three colored boxes, click on the box.  You may also press the UP ARROW key to move the card to the "Know" box, the DOWN ARROW key to move the card to the "Don't know" box, or the RIGHT ARROW key to move the card to the Remaining box.  You may also click on the card displayed in any of the three boxes to bring that card back to the center.

Pass complete!

"Know" box contains:
Time elapsed:
Retries:
restart all cards




share
Embed Code - If you would like this activity on your web page, copy the script below and paste it into your web page.

  Normal Size     Small Size show me how

BIOL Exam I - Ex. 3

Advanced Techniques of Light Microscopy

QuestionAnswer
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
Created by: jessica.gvc