Visualizing Cells + Manipulation of Cells (ch 8, 9) Flashcards
What is the limit of resolution for the naked eye? What about light microscopes? Electron microscopes?
Eye: ~0.5 mm
Light microscope: ~100nm
Electron microscope: ~0.1 nm
Who coined the term “microscope”?
Giovanni Faber.
What is meant by “optical diffraction”?
Two light waves out of phase can limit the resolution of a microscope because of interference effects.
Using light microscopy, when do we reach the limit of resolution for two points close together?
When the points are less than 200 nm apart.
Resolution is dependent on 2 factors. What are they?
- Wavelength
2. Numerical aperture
How can we calculate the resolution of a microscope?
= 0.61λ / nsinθ Where: n is the index of refraction θ is the angle of incidence λ is the wavelength
What is the purpose of the objective lens in a light microscope?
Collects a cone of light rays to form an image.
What is the purpose of the condenser in a light microscope?
Focuses a cone of light rays onto each point of the specimen.
What differentiates resolution and detection?
Resolution: discern details about the specimen
Detection: discern the presence of a subject
What is the resolution given n=1, λ=530nm, θ=20?
~950 nm.
What are the 4 basic types of light microscope?
- Bright field
- Dark field
- Phase contrast
- Differential-interference contrast
What is the main problem with bright field microscopy? How can we fix this? What drawback exists?
Specimens are grey-on-grey with no contrast. Can use dyes or stains but have to fix (kill) cells to add colour.
How does dark field microscopy work?
Prevent direct light from entering objective, only allow scattered light.
How does phase contrast microscopy work?
Waves out of phase generate contrast when combined. The phase shift causes the change in brightness.
How does differential-interference contrast microscopy work?
Like phase contrast microscopy, but uses polarized light to give the appearance of a 3D image.
How can we detect small objects that are below the resolution limit of light microscopes?
- Electron microscopy
2. Fluorescence microscopy (labelling)
In fluorescence microscopy, electrons are _____ at a shorter wavelength and ______ at a longer wavelength.
Excited at shorter wavelength, emitted at longer wavelength.
Which fluorescent dye is used to stain all DNA?
DAPI.
Which fluorescent dye is used to stain all proteins?
FITC.
Which 4 fluorescently labelled antibodies were shown in lecture?
- Rhodamine B
- Alexa 568
- Cy5
- Cy3
Which 4 fluorescent proteins are often used in genetic engineering?
- GFP
- CFP
- YFP
- RFP
What is “autofluorescence”? What is a downside of this?
A phenomenon where cells can be excited to fluoresce at a specific wavelength. Can interfere with added fluorescence.
What are the 3 main elements of a fluorescent microscope? What light does each part interact with?
- 1st barrier filter: passes blue
- Beam-splitting mirror: reflects blue, passes green
- 2nd barrier filter: passes specific green
How does confocal fluorescence microscopy work? How can it be used?
Detects only the in-focus light which passes through a pinhole. Can be used to make sections which can be combined to form a 3D image.
What 3 strategies can we use to detect specific molecules in a microscopy sample?
- Dyes/stains
- Antibodies
- Genetic engineering: fluorescent proteins
When would you want to use a dye instead of a different identifier (antibodies, GFP, etc.)?
When you want to detect all of a specific class of molecule (ex: DNA).
What is most often used as a primary antibody?
Rabbit antibody.
What is most often used as a secondary antibody?
Sheep antibody.
How do fluorescent-tagged antibodies work?
1º antibody detects antigen “A” on cell surface. 2º antibody with fluorescent marker binds to 1º antibody.
Do all organisms produce antibodies?
No. Only animals, and more specifically vertebrates, make antibodies.
How can antibodies be used to identify DNA at centromeres?
By targeting the histone proteins that are centromere-specific.
How can GFP be used as a reporter sequence?
By adding it into a gene, we will observe GFP fluorescence every time that gene is expressed.
What strategy can we use to determine intracellular localization of a synthesized protein?
Insert a GFP sequence into the gene so that synthesized protein has fused GFP and fluoresces wherever it ends up.
What is photoactivated GFP? Why might this be used?
GFP which must be activated by light in a certain area. Can be used to visualize protein diffusion.
What is photobleaching? Why might this be used?
Permanent deacivation of fluorescence. Can be used to visualize protein mobility/diffusion in the cell.
Briefly describe fluorescence resonance energy transfer (FRET).
Protein interaction can be identified depending on the colour of light which is emitted by the fluorescent proteins.
Why might we use total internal reflection (TIRF) microscopy?
Gives a crisp image which shows only the surface molecules. Other molecules are invisible.
How come electron microscopes have better resolution than light microscopes?
Electrons have a shorter wavelength than light.
What are some limitations/disadvantages to electron microscopy?
Specimen must be dead, can only observe in a vacuum, specimens must undergo complex preparation.
How do we prepare specimens for electron microscopy? (4 steps)
- Fixation (glutaraldehyde or osmium tetroxide)
- Dehydration
- Permeation with resin
- Slice into thin sections
How can we label samples for electron microscopy?
Not with fluorescence but instead with gold. Density of gold reflects electrons differently to allow contrast.
What are some specific characteristics of scanning electron microscopy?
- Cheaper than tEM
- Looks at surface anatomy only
- Must coat w/ gold layer otherwise no contrast
- specimens frozen and dehydrated
- vacuum chamber required
What is the scanning transmission electron holographic microscope (STEHM)?
Built at UVic, can create a holographic image of a subject. Best resolution of any microscope. Combines EM with computer tomography.
How can we isolate a specific cell from a tissue? What 2 steps are involved?
- Disrupt extracellular matrix w/ enzymes or EDTA
2. Sort cell types w/ FACS based on charge
What is an FACS?
Fluorescence activate cell sorter.
What is an alternative way to separate specific cells from tissue without using FACS?
Laser capture microdissection.
How can we isolate organelles from cells?
By centrifugation at progressively increasing speeds.
What organelles will precipitate when centrifuged at low speed?
Whole cells, nuclei, cytoskeletons.
What organelles will precipitate when centrifuged at medium speed?
Mitochondria, lysosomes, peroxisomes(/chloroplasts).
What organelles will precipitate when centrifuged at high speed?
Microsomes, small vesicles.
What organelles will precipitate when centrifuged at very high speed?
Ribosomes, viruses, large macromolecules.
How can proteins be isolated from cells?
Column chromatography.
What are the 3 types of column chromatography, ordered by size of molecule isolated (largest to smallest)?
- Ion-exchange chromatography
- Gel-filtration chromatography
- Affinity chromatography
Describe ion-exchange chromatography.
Positively charged beads in column bind negatively charged molecules.
Describe gel-filtration chromatography.
Porous beads in column capture small molecules and allow large ones to pass.
Describe affinity chromatography.
Beads in column have attached substrate and so capture enzymes.
What was the “neuronal doctrine” argument?
The early 20th cent. disagreement between the idea that 1 cell could grow into a tissue vs the idea that many cells fused to make a tissue.
What differentiates a 1º culture from a 2º culture?
1º: cells derived from the original donor tissue
2º: re-cultured cells in new colony.
In cell biology, how do we define a cell culture described as being “in-vitro”?
Parts of cells or fractions in a test tube.
In cell biology, how do we define a cell culture described as being “in-vivo”?
Regards whole cells, even those which are grown in a dish (different from medical defn.).
What human cell line is commonly used for cell cultures? Where does this cell line come from?
HeLa cells. Grown from tumour cells taken from Henrietta Lacks (against her knowledge).
What is meant by “replicative cell senescence”?
Cells can only replicate a limited number of times before they are programmed to die.
What is the advantage of replicative cell senescence?
Helps to reduce the risk of cancer and unwanted mutations.
What replication machinery (related to senescence) do many prokaryotes and other organisms have that human cells lack?
Telomerases which can elongate telomeres to preserve replicative ability.
What is a hybridoma cell? Why use them?
A fusion of cells from 2 different organisms often used for antibody production.
What is one major application of hybrid cell use?
Gene mapping.
What does CRISPR stand for?
Clustered regularly interspaced short palindromic repeats.
How can CRISPR-Cas9 be used?
For gene-editing.
In CRISPR, what is the purpose of the Cas9?
To cause a double strand break, opening up the genome for transgenic gene integration.
How can Cas9 be modified in CRISPR?
With an activation or repression domain so that it can influence gene expression instead of causing a double strand break.
