Exam 1 Flashcards
What are the three main branches of microscopy?
Optical (light, or bright field) microscopy, electron microscopy, and scanning probe microscopy.
What defines optical (light, or bright field) microscopy?
This type of microscopy uses light in the visible light spectrum that transmits through or reflects from a microscopic sample.
What are the major limitations of optical (light, or bright field) microscopy?
This type of microscopy can only show dark objects
Diffraction limits the resolution to a size of 0.2 micrometers
Points outside the image plane are blurry
What is the difference between magnification and resolution?
Magnification is how big something is, and resolution is how detailed you can see something.
What does bright field microscopy do?
It detects what parts of specimen absorb light, so it looks dark against a light background
What can you do in light microscopy if the part of the cell you want to see is transparent? What are the downsides to this solution?
You can use specific stains that bind to specific structures of interest in the cell.
The dye could kill the cell or change the performance or behavior of the cell that you are investigating
The dye could stain things other than what you are trying to stain
The dyes are chemicals, not antibodies, so they are not that specific
Dye might not go through the cell membrane since it is large and or charged so you might have to fix the cell and then slice it into sections so the dye can get access the cell
What does phase contrast microscopy do?
This technique shows differences in refractive index (result of slowing of the light wavefront as it passes through a transparent object) as difference in contrast by converting differences in refractive index in the specimen into differences of intensity in the image. This requires no stain.
What does DIC (differential interference contrast / Nomarski) microscopy do?
This technique detects objects in which the gradient of refractive index change is greatest, and thus is good at amplifying the edges of organelles (ex: membrane and edge of organelles). This does not require stain.
What does polarization microscopy do?
With this technique the microscope selects for certain changes in the plane of polarization. When light passes through a structure it can be polarized, and some structures shift the plane of polarization, and the microscopes looks for these specific changes. This does not use dye and it is good for identifying some highly ordered “crystalline” structures.
What does fluorescence microscopy do?
Fluorescence microscopy detects fluorescent molecules. The specimen is illuminated with intense light of one wavelength. The fluorescent objects absorb this excitation light and emit another, longer wavelength of light (always longer). This emitted light detected by microscope using color filter in the microscope that pass only the emitted light. Some cell structures are naturally fluorescent.
How can fluorescent markers take advantage of fluorescence microscopy?
Artificial dyes that are “fluorescent markers” can be coupled to antibodies that allow for imaging of specific proteins in cells. Usually, but not always, slices of fixed, dead cells are used so antibodies get maximum access to the proteins. These target proteins, thus, flouress.
What is the difference between luminescence and fluorescence?
Luminescence produces light, while fluorescence requires light to show fluorescence.
What are the downsides of fluorescence microscopy?
In order for large hydrophilic proteins (such as antibodies) to access proteins inside cells, cells have to be chemically fixed or frozen and sectioned (so they are dead).
What does fluorescence energy transfer (FRET) microscopy do?
It detects when two specially designed fluorescent
molecules come within a nanometer or so of each
other. Can be used to detect movement of functional groups within the molecule or the binding of one protein to another.
What does confocal microscopy do?
This method involves using a scanning point of light instead of full sample illumination. This microscopy gives slightly higher resolution, and significant improvements in optical sectioning. Confocal microscopy is, therefore, commonly used where 3D structure is important.
What does total internal reflection fluorescence microscopy do?
This technique can be used to detect single “tagged” molecules in a specimen on a cover slip (but cannot resolve their structures). Light comes in at an acute angle and all of the light is reflected off of the coverslip but some of the light energy extends past the cover slip which can excite fluorescence from the molecules at the top of the coverslip. This large magnification but not great resolution.
What does super-resoultion fluorescence microscopy do?
This technique allows the capture of images with a higher resolution than the light diffraction limit (200 - 300 nm)
What does stimulated emission depletion (STED) microscopy do?
This technique uses special fluorescent dyes that can be excited by a pulse of light of one wavelength and quenched (fluorescence is prevented) by a second pulse of light of another wavelength. The change in light needs to occur very quickly so it can be excited and quenched so quickly that the emitted spot of fluorescence is very
small and the resolution can be precise. This can be used to see the tertiary structure of proteins.
What does near-field scanning fluorescence microscopy do?
dispenses with the objective lens and instead forces the exciting laser light down a tiny nanometer-diameter optical fiber on to a fluorescent specimen. If this fiber optic cable is place a few nanometers above the specimen surface, light from it will be restricted to a spread of just a few tens of nanometers across the surface - so called “near field” effect microscopy. Fluorescent light emitted from this spot can be collected by a conventional lens as the optical fiber is moved across the specimen surface.
What does atomic force microscopy do?
This method is a non-optical method in which a tiny probe tip is dragged across the surface of a specimen. It is especially useful in examining the surface of biological membranes with nanometer resolution. By monitoring the distances over which the probe is raised and lowered as it scans the surface, a topological map of the surface structure can be created and an image of the surface can be generated.
What does electron microscopy do?
With this method, an electron beam is focused by magnets in vacuum, viewed by phosphor screen or film. Tissue must be dead, fixed, embedded in plastic, dehydrated, and stained.
In general, what is a T.E.M. technique?
Instead of staining the specimen, you stain the stuff the specimen is in so everything around the thing you want to see is dark so you can see what you want. it is like reverse staining or “negative
staining
What does freeze fracture T.E.M. do?
Freeze something and hit it with something hard and look at how the crack propagates. Cells usually crack between the leaflets of cell membrane (between the layers of the bilayer). The results look like the surface of the moon (because of a shadowing effect)
What does a scanning electron microscope do?
Tissue is fixed, coated with electron dense material but is not sectioned. It detects secondary electrons emitted as electron beam sweeps across
surface of specimen and builds up a “3D” image.
List 5 universal features of cells
- Store genetic information as linear DNA
- Transcribe portions of hereditary information as some intermediary form
- Use proteins as catalysts
- Replicate hereditary information as template polymerization
- Translate RNA to protein to express genes
- Made up of nucleic acids, lipids, and carbohydrates
- Enclosed in plasma membrane composed of a lipid bilayer and membrane proteins
- Require a source of free energy to function
Explain the subdivisions of homologs
Orthologs arise from divergence through speciation
(Usually serve same function)
Paralogs arise from gene duplication within a species / organism
(Have new but related functions)
What are the criterions for model organisms? What are 5 current model organisms?
- Entire genome has been sequenced
- Site-directed and tissue-specific mutagens exist
- Expression of multiple genes can be tracked across many cells simultaneously
- Developmental sequence is known
Arabadopsis, mouse, c. elegans, drosophila melanogaster, yeast
What are 5 ways cells can do work?
- Electrical (separation of charges)
- Mechanical (moving things around)
- Producing light
- Producing heat
- Making and breaking chemical bonds
What are three ways cells store energy?
- Energy in bonds
- Energy in electrochemical potential
- Energy in chemical concentration gradients
How can cells decrease entropy?
•By forming macromolecules
Describe anabolic and catabolic reactions, and how they relate to free energy.
- Anabolic reactions involve the synthesis of complex molecules
- Catabolic reactions involve the break down of complex molecules
Often, catabolic reactions are coupled to anabolic reactions which provides the anabolic reaction some free energy
What is the difference between equilibrium and steady states? What is a living cell in?
•Equilibrium means that there is a constant rate of products to reactants and reactants to products, and there it is a closed system
•Steady state means that there is an input of energy on the reactants side and an output of energy on the reactants side, but the concentrations of products and reactants stay the same
(There is a steady net flux of materials through coupled reactions which requires a constant input of energy)
•Living cells are in a steady state and dead cells are in equilibrium
What are the players in redox reactions?
•Redox reactions involved something getting oxidized and something getting reduced.
Something is reduced when electrons are added to it
Something is oxidized when electrons are taken from it
Something is oxidized when oxygen or nitrogen is added to it
Something is reduced when oxygen or nitrogen is taken from it
Why is the oxidation of hydrocarbons so lucrative?
When a H bonds to O2, this reaction is very energetically favorable. This reaction has a very negative delta G, and its product, H2O, is very stable and has low energy. Therefore it is lucrative for the body to strip hydrocarbons of their H’s and bind them with O2 so the body can gain some free energy to do work elsewhere
When one molecule of glucose goes through glycolysis in the presence of oxygen, what are the products? What about in the absence of oxygen?
- Glycolysis – 2 ATP, 2 Pyruvate, and 2 NADH
- With fermentation, the cell uses NADH to reduce pyruvate and regenerate NAD+ in the cytosol
This keeps NADH from building up in the cell which would be bad because this would increase the concentration of products so the reaction would not drive forward so much
Why does fat store so much energy?
•The long hydrocarbon tail bonded to glycerol stores bonds that can be broken and H’s and C’s that can be bound to O2 which releases tons of energy making CO2 and H2o
How is fat turned into useable energy?
- Fat is broken down into fatty acids and glycerol. Fatty acids are transported across the cell membrane and go into the blood stream. The fatty acids flow through adipose tissue and can then be delivered to muscle cells. In muscle cells, the hydrocarbon tails of the fatty acids are oxidized into CO2
- Fatty acids can also link to CoA like pyruvate and can go through the Krebs cycle, producing FADH and NADH. This happens in mitochondrial metabolism.
When fatty acid goes through the Krebs cycle, it continuously becomes 2 carbons shorter and will go through cycle until it is done.
How are amino acids made and broken down?
- The products of glycolysis and the Krebs cycle are building blocks for amino acids
- Amino acids can be broken down to help for acetyl CoA
What is a residue?
•The remains of covalently bound amino acids are residues
What determines the percent of residues that are actually charge (or the probability that a residue is charged)?
•The probability a residue is charged is based on the pH of the solution it is in
What determines a protein’s secondary through quaternary structure?
•The protein’s primary structure based on the original mRNA code and the protein’s specific environment
What bonds are responsible for holding a protein’s secondary structure together? What are the two most common secondary structures?
- The secondary structure is held together by weak H bonds between the C-O (carbonyl) groups and N-H (amine) groups in the backbone. NOT the R groups.
- Alpha helices and beta sheets
What bonds are responsible for holding a protein’s tertiary structure together? What are the two most common tertiary structures? Why do the proteins fold into tertiary structures?
- The tertiary structure is mostly held together by weak bonds between R groups and the surrounding environment (like the bilayer interior the protein is in).
- The tertiary structures of proteins usually arise from self-folding into the most low energy structure, but sometimes enzymes called chaperonins catalyzing the folding.
What are the two exception that introduces covalent bonding which effects secondary and tertiary structure in protein folding?
- Rigid proline residues insert a kink into a protein’s backbone which disrupts a protein’s secondary structure
- Covalent disulfide bonds can form between cysteine residues to cross-link parts of the polypeptide backbone
How can you determine definitive tertiary structures of proteins?
•Using x-ray crystallography or protein crystals grown in-vitro from purified protein you can see the tertiary structure
What are the differences between Myoglobin and Bacteriorhodopsin?
- Myoglobin is a soluble protein mostly made of alpha helical domains and is stable in solutions
- Bacteriorhodopsin is an integral membrane protein mostly made of alpha helices and is stable in membranes
What is a domain and what are some examples of domains?
•A domain is a structural unit within a protein’s sequence – can either be described by structure or function
Alpha helical domains, coiled-coiled domains (alpha helices coiled around one another), Beta sheet domains, random coil domains (no secondary structures), catalytic domain, ligand binding domain
What is an example of a protein with multiple domains?
•The myosin motor protein has an alpha helical domain and a globular domain
What is a motif?
•A motif is a similar domain is many related proteins (like DNA binding motif). Can be defined according to function
What bonds hold together quaternary structure?
•Held together by weak bonds and some disulfide bonds
What is the difference between a homomer and a heteromer?
- A homomer is an identical polypeptide subunit
* A heteromer is different polypeptide subunits
What are protein complexes and what are they for?
- Protein complexes are formed by protein protein interactions. Binding domains on some proteins bind to domains other proteins through weak bonds.
- Sometimes, this is used when the produce of one enzyme to be rapidly transferred to the substrate binding site of another enzyme. Also it can be used to tether a group of proteins to a specific site in a cell.
What drives conformation changes in the tertiary structure of proteins, and how quickly can it happen?
- Fluctuations in thermal (kinetic) energy drives spontaneous conformational changes
- Large changes may take microseconds, and small changes may take nanoseconds
What are ligands and how are they bound?
- Ligands are molecules proteins bind to. For enzymes these are called substrates.
- Ligand binding is achieved by weak bonds (not covalent) so it is reversible
- The amino acids residues that contribute to binding a ligand are often far apart of the protein’s primary structure but when the protein is all folded up, they are close enough to help with the ligand binding.
- There is a dynamic equilibrium between the binding of a protein to the ligand
What kinds of protein mutations are there? What is an example of a single site mutation?
- Conservative mutations – polar for polar etc…
- Non-conservative mutations – polar for charged etc…
- Mutations can be random or genetically engineered
- Sickle cell disease has one amino acid change in oxygen-binding protein
What is site-directed mutagenesis?
•cDNA coding for a protein is amplified by cloning it in bacterial cells and mutation in vitro and a genetically modified organism is made
What are the three postulates of 19th century cell theory, and which one turned out to be wrong?
- All living organisms are composed of nucleated cells (wrong)
- Cells are the functional units of life
- Cells arise only from pre-existing cells by a process of division
What is the fewest number of genes a cell can function?
500 genes.
What sources of delta G can cells be powered with, and what are those types of cells / organisms called?
Light - phototrophs
Organic chemicals - organotrophs
Inorganic chemicals - lithotrophs
