Topic 2 - Working with Cells Flashcards
How small of images can the human eye see, a light microscope, and an electron microscope?
- 10^-4 m
- 10^-7 m
- 10^-9 m or 1 picometer
Name one or two different objects that can be identified with the naked eye, light microscope, and an electron microscope?
- frog egg, larvae
- cells, bacterium
- atom, globular proteins, virus ribosomes
What is the conventional resolution limit of a light microscope?
- up to 200nm (40X magnification – but can be extended to 1000X with oil immersion)
Explain the challenges of interference effects
- trying to observe wavelengths in different phases may allow the light to appear BRIGHT or DIM
When is the limit of resolution reached (2)
- when two points are no longer distinguishable
- when one point starts to appear as a blurry disk
Resolution definition
- is the shortest distance between two points that can sill be distinguished between
What are the two things that resolution is dependent on?, and what is the equation?
- wavelength and numerical aperture (n X sin theta)
- resolution = (o.61 X wavelength) / (n X sin theta)
Distinguish between resolution and detection
Resolution - looks at how clear the image is that you are looking at
Detection - how big or blurry the image is (the presence or absence of the image)
What are the 4 basic types of microscopes?
- Bright field microscope
- Dark field microscope
- phase contrast microscope
- differential-interference contrast microscope
The pros and cons of bright field microscopy.
- lacks biological detail in colour and contrast
- able to stain the sample but this requires killing, preserving or immobilizing the cells – so now you cannot view the cellular machinery
Explain what happens in dark-field microscopy, and one example is it used for?
- an opaque disk attempts to filter out any direct light so we only observe the scattered light that went through the sample on a black background
- radiolora
Explain the workings of Phase-contrast microscopy
- when you observe the sample you are looking at light waves traveling at different amplitudes as it moved through the sample – able to observe the brightness
Explain how differential-interference microscopy works
- polarized light (light in one plane) is used to produce a 3D image
Give two examples of fluorescent dyes and what they would be used for.
- DAPI: binding to DNA as a whole
- FITC: which can bind to proteins
Give two more methods used for Fluorescent Microscopy
- fluorescently labelled antibodies
- fluorescent proteins which are genetically engineered
What is autofluorescence?
- small molecules which have fluorescent ability such as NADH
Confocal Fluorescent Microscopy produces what sort of image?
3D fluorescent images
What molecules can be detected using dyes, antibodies, or GFP proteins?
- DAPI dyes to detect DNA (Note: FITC also a dye but for proteins)
- Antibodies will detect VERY specific proteins or transcription factors
- GFP will detect gene expression factors, protein turn over rates & protein localization
Explain how dyes work, and their one short coming
Dyes are used to detect DNA as a whole, they cannot be used to detect specific chromosomes (this requires specific labeling)
When do you use fluorescent tagged antibodies?
Fluorescently tagged antibodies would be used to target very SPECIFIC proteins
How does antibody production work for specific proteins?
- first would require isolation of the protein/antigen A
- this protein A is injected into a rabbit who will produce its own antibodies for protein A
- now fluorescently produced secondary antibodies which are labelled which are targeted for the specific rabbit antibody will be performed (secondary antibody production from a goat?)
- this means the primary rabbit antibody binds the protein A but then the labeled secondary antibody from the goat will bind the rabbit antibody and the specific protein A will be detected
- cheaper process and they are already labelled
How can you combine methods for multi-fluorescent -probe microscopy?
The use of green fluorescent antibody for spindle microtubules, red fluorescent antibody for the centromere, and DAPI (fluorescent dye) for chromosomes
What can we detect with genetically engineered fusion proteins? (cis-regulated DNA sequence)
we detect a glow when our new sequence is translated and expressed – further this allows us to determine in which cell cycle stage the cell is and where it is expressed in the cell
What are the key factors in performing the genetically engineered fusion proteins? (cis-regulated DNA sequence)
through recombination, you insert the GFP protein construct for reporter protein Y which uses the same normal cis-regulating DNA sequence for the expression of gene X and protein X`
How can we perform truncated experiments.
By genetically engineered fusion proteins that allows us to observe the expression and turning on and off of genes and how genes are regulated.
What is signal peptide marker gene fusion? (signal peptide marker) and what do we see?
NOTE: no promoter sequence
- the presence of the peptide end terminus (which directs the protein where to go)
- this allows intracellular localization
What is protein marker gene fusion? (protein marker) and what do we see?
- this method allows for protein-protein interaction and protein turn over rate
What is photo-activation? and what is it used for?
- the protein will only fluoresce after photo-activation by a fluorescent light
- this is used to determine how fast the protein activates and where it moves to within a cell
What does FRAP stand for? and how is this method down
Fluorescent Recovery after Photo-bleaching
- GFP fused to a protein will be photo-bleached
- the fluorescent signal is destroyed
- then you wait for the recovery of the protein by new expressions
What does FRET stand for? and what do we learn from this method?
Fluorescent resonance energy transfer
- protein-protein interactions
- must see Lecture Questions for explanation
What does TIRF stand for? and what do we observe with this technique?
Total Internal Reflection Fluorescence
- this is used when you only want to observe certain SURFACE proteins by canceling out the background
what is the magnification ability difference between light and electron microscopes
200nm vs 1nm respectively
How can electron microscopy improve resolution
- No longer use light - electron wavelength are much shorter
What is the purpose of the vacuum in an EM?
any dust or particles in the air will collide with the electrons you observe
What is an advantage to EM?
observations are 200X better
What is a disadvantage to EM?
- expensive
- complex species preparation
- proteins need to be fixed to prevent denaturing or rearrangement (dehydrate the sample)
- fixing components are: gluteraldehyde and osmium tetroxide
- samples are cut into thin slices
What can you observe with an EM?
- cell wall
- golgi
- nucleus
- mitochondrion
- ribosomes
What can you observe with immunogold EM? and how is this done
- individual proteins and primary and secondary antibodies
- sample is labelled with colloidal gold particles
What are some key points of Scanning EM?
- the specimen is dried or frozen
- specimen will be labelled with a thin layer of a heavy metal or gold
- has a resolution of 10nm
- the sample is bombarded with electrons which are scattered and then collected on a detector
STEHM stands for what?
- Scanning Transmission Electron Holographic Microscope
- best resolution microscope ever built: up to 40 pm (which is the diameter of a helium atom)
- measure both phase and amplitude of the material
- determines the species absolute composition
What does labelling molecules allow you to do with light microscopy? and what does EM allow us to do?
- allows the detection of molecules below the resolution limit
- it allows us the detailed analysis of intracellular organization and surfaces
What are two steps required to isolate specific cell types from a tissue?
- EDTA is used to chelate Ca2+ that a cell naturally uses for cell-cell interactions
- using proteolytic digesting to disrupt the ECM and any cell-cell interactions
Why would you want to isolate specific cell types
To characterize the differences between a tumour cell and a healthy cell (metabolites and proteins - drug testing)
In short, quickly explain FACS
Fluorescent Activated Cell Sorter
- selects up to 1000cells/second
- a container with a cell suspension and cancer cells which are labelled are dropped into specific tubes
- a laser and defector will determine if a fluorescent cell will receive either a positive or negative charge as it drops down
- once the receive their charge they pass through a voltage tube where they are separated into the respective tube
What is Laser Capture Micro-Dissection
cells are placed on a microscope slide covered in plastic, then observing under a microscope the selected sample is excised with a laser
How do you isolate and purify organelles?
Homogenize the cells via centrifugation at different speeds, each time the supernatant will have the finer organelles
Proteins can be separated through these 4 techniques and a fluorescently labelled type of way?
- Column chromatography
- Ion-exchange
- Gel Filtration
- Affinity Chromatography
- the gene of interest has an epitope tag added through recombination which can be purified out
What is a homogenous cell population and what is the difference between primary and secondary cultures
- cell cultures that can be frozen to allow repeat experiments
- primary cultures were directly isolated from the organism while secondary cultures are re-cultured by transferring it to new media
What is the difference between in vivo and in vitro?
- living system vs in a glass
What are key features of Human and Plant cells
- they require solid surfaces to grow and do not grow on top of themselves (one layer only)
- plant tissue can produce a calli* to regenerate itself
Define Replicative Senescence
normal cells will only replicate for about 25-40X before entering senescence
What are two reasons behind replicative senescence?
- telomerase shortening
- replicative check points
What are hybrid cells and what can their function be applied to?
- it is a cell fusion product where one partner was a tumour cell
- these cells can be used to ensure the same target antibody is produced everytime
What are transgenic organisms capable of?
using an expression vector, a protein-coding DNA sequence is inserted in front of a promoter sequence and through recombinant DNA the cell will over express the mRNA for the desired protein
Give two types of mutations that can occur causing transgenic organisms
- LOF mutation: point mutation, deletion, truncation
- conditional LOF: temperature sensitive
Define CRISPR
- cluster regularly interspaced short palindromic repeats
- is a site specific manipulation of a genome (used by bacteria on viruses)
Explain how CRISPR works
- viral DNA is inserted into a bacteria cell where short segments are cleaved
- these segments are inserted into the CRISPR locus
- the RNA from the CRISPR locus are transcribed and attached to the Cas proteins (crRNAs)
- these crRNAs and the Cas9 protein will see out and destroy the viral sequences if they ever return
How is the Cas9 protein so effective?
it functions as an endonuclease that is guided by mRNA matches that are positioned in the genome
Transgenic organisms - as plants
The DNA is excised from plasmids as a linear molecule, transferred directly into the plant cell where it become integrated into the plant chromosome
