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Bio 360 - Cell Biology > Topic 2 - Working with Cells > Flashcards

Topic 2 - Working with Cells Flashcards

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1
Q

How small of images can the human eye see, a light microscope, and an electron microscope?

A
  • 10^-4 m
  • 10^-7 m
  • 10^-9 m or 1 picometer
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2
Q

Name one or two different objects that can be identified with the naked eye, light microscope, and an electron microscope?

A
  • frog egg, larvae
  • cells, bacterium
  • atom, globular proteins, virus ribosomes
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3
Q

What is the conventional resolution limit of a light microscope?

A
  • up to 200nm (40X magnification – but can be extended to 1000X with oil immersion)
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4
Q

Explain the challenges of interference effects

A
  • trying to observe wavelengths in different phases may allow the light to appear BRIGHT or DIM
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5
Q

When is the limit of resolution reached (2)

A
  • when two points are no longer distinguishable

- when one point starts to appear as a blurry disk

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6
Q

Resolution definition

A
  • is the shortest distance between two points that can sill be distinguished between
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7
Q

What are the two things that resolution is dependent on?, and what is the equation?

A
  • wavelength and numerical aperture (n X sin theta)

- resolution = (o.61 X wavelength) / (n X sin theta)

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8
Q

Distinguish between resolution and detection

A

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)

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9
Q

What are the 4 basic types of microscopes?

A
  • Bright field microscope
  • Dark field microscope
  • phase contrast microscope
  • differential-interference contrast microscope
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10
Q

The pros and cons of bright field microscopy.

A
  • 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
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11
Q

Explain what happens in dark-field microscopy, and one example is it used for?

A
  • 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
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12
Q

Explain the workings of Phase-contrast microscopy

A
  • 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
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13
Q

Explain how differential-interference microscopy works

A
  • polarized light (light in one plane) is used to produce a 3D image
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14
Q

Give two examples of fluorescent dyes and what they would be used for.

A
  • DAPI: binding to DNA as a whole

- FITC: which can bind to proteins

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15
Q

Give two more methods used for Fluorescent Microscopy

A
  • fluorescently labelled antibodies

- fluorescent proteins which are genetically engineered

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16
Q

What is autofluorescence?

A
  • small molecules which have fluorescent ability such as NADH
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17
Q

Confocal Fluorescent Microscopy produces what sort of image?

A

3D fluorescent images

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18
Q

What molecules can be detected using dyes, antibodies, or GFP proteins?

A
  • 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
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19
Q

Explain how dyes work, and their one short coming

A

Dyes are used to detect DNA as a whole, they cannot be used to detect specific chromosomes (this requires specific labeling)

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20
Q

When do you use fluorescent tagged antibodies?

A

Fluorescently tagged antibodies would be used to target very SPECIFIC proteins

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21
Q

How does antibody production work for specific proteins?

A
  • 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
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22
Q

How can you combine methods for multi-fluorescent -probe microscopy?

A

The use of green fluorescent antibody for spindle microtubules, red fluorescent antibody for the centromere, and DAPI (fluorescent dye) for chromosomes

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23
Q

What can we detect with genetically engineered fusion proteins? (cis-regulated DNA sequence)

A

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

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24
Q

What are the key factors in performing the genetically engineered fusion proteins? (cis-regulated DNA sequence)

A

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`

25
Q

How can we perform truncated experiments.

A

By genetically engineered fusion proteins that allows us to observe the expression and turning on and off of genes and how genes are regulated.

26
Q

What is signal peptide marker gene fusion? (signal peptide marker) and what do we see?

A

NOTE: no promoter sequence

  • the presence of the peptide end terminus (which directs the protein where to go)
  • this allows intracellular localization
27
Q

What is protein marker gene fusion? (protein marker) and what do we see?

A
  • this method allows for protein-protein interaction and protein turn over rate
28
Q

What is photo-activation? and what is it used for?

A
  • 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
29
Q

What does FRAP stand for? and how is this method down

A

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
30
Q

What does FRET stand for? and what do we learn from this method?

A

Fluorescent resonance energy transfer

  • protein-protein interactions
  • must see Lecture Questions for explanation
31
Q

What does TIRF stand for? and what do we observe with this technique?

A

Total Internal Reflection Fluorescence

- this is used when you only want to observe certain SURFACE proteins by canceling out the background

32
Q

what is the magnification ability difference between light and electron microscopes

A

200nm vs 1nm respectively

33
Q

How can electron microscopy improve resolution

A
  • No longer use light - electron wavelength are much shorter
34
Q

What is the purpose of the vacuum in an EM?

A

any dust or particles in the air will collide with the electrons you observe

35
Q

What is an advantage to EM?

A

observations are 200X better

36
Q

What is a disadvantage to EM?

A
  • 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
37
Q

What can you observe with an EM?

A
  • cell wall
  • golgi
  • nucleus
  • mitochondrion
  • ribosomes
38
Q

What can you observe with immunogold EM? and how is this done

A
  • individual proteins and primary and secondary antibodies

- sample is labelled with colloidal gold particles

39
Q

What are some key points of Scanning EM?

A
  • 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
40
Q

STEHM stands for what?

A
  • 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
41
Q

What does labelling molecules allow you to do with light microscopy? and what does EM allow us to do?

A
  • allows the detection of molecules below the resolution limit
  • it allows us the detailed analysis of intracellular organization and surfaces
42
Q

What are two steps required to isolate specific cell types from a tissue?

A
  • 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
43
Q

Why would you want to isolate specific cell types

A

To characterize the differences between a tumour cell and a healthy cell (metabolites and proteins - drug testing)

44
Q

In short, quickly explain FACS

A

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
45
Q

What is Laser Capture Micro-Dissection

A

cells are placed on a microscope slide covered in plastic, then observing under a microscope the selected sample is excised with a laser

46
Q

How do you isolate and purify organelles?

A

Homogenize the cells via centrifugation at different speeds, each time the supernatant will have the finer organelles

47
Q

Proteins can be separated through these 4 techniques and a fluorescently labelled type of way?

A
  • Column chromatography
  • Ion-exchange
  • Gel Filtration
  • Affinity Chromatography
  • the gene of interest has an epitope tag added through recombination which can be purified out
48
Q

What is a homogenous cell population and what is the difference between primary and secondary cultures

A
  • 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
49
Q

What is the difference between in vivo and in vitro?

A
  • living system vs in a glass
50
Q

What are key features of Human and Plant cells

A
  • 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
51
Q

Define Replicative Senescence

A

normal cells will only replicate for about 25-40X before entering senescence

52
Q

What are two reasons behind replicative senescence?

A
  • telomerase shortening

- replicative check points

53
Q

What are hybrid cells and what can their function be applied to?

A
  • 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
54
Q

What are transgenic organisms capable of?

A

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

55
Q

Give two types of mutations that can occur causing transgenic organisms

A
  • LOF mutation: point mutation, deletion, truncation

- conditional LOF: temperature sensitive

56
Q

Define CRISPR

A
  • cluster regularly interspaced short palindromic repeats

- is a site specific manipulation of a genome (used by bacteria on viruses)

57
Q

Explain how CRISPR works

A
  • 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
58
Q

How is the Cas9 protein so effective?

A

it functions as an endonuclease that is guided by mRNA matches that are positioned in the genome

59
Q

Transgenic organisms - as plants

A

The DNA is excised from plasmids as a linear molecule, transferred directly into the plant cell where it become integrated into the plant chromosome

Bio 360 - Cell Biology (10 decks)

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